in food processing plants , residues of all kinds chemical , biological , organic , or inorganic inevitably accumulate on the surfaces of equipments in contact with food .   attachment of undesirable microorganisms to these surfaces is a source of concern , since this can result in product contamination leading to serious economic and health problems [ 24 ] .  
in fact , this microbial contamination has two components : first , the saprophytic flora responsible for food spoilage and second , the pathogenic flora , which cause infections in humans and animals .   to adversely affect the sensory , physical , and chemical qualities of food , a large population of spoilage - causing microorganisms is required , while in the case of food pathogens it only takes a few cells to affect product safety and cause food poisoning . in the phenomenon of bacterial adhesion to inert surfaces ,
the physicochemical properties ( hydrophobicity and charges ) and substrates or surface topography are playing important roles [ 57 ] .
joints such as valves and any other difficult - to - reach spaces are the most favourable areas to bacterial adhesion . the effect of corrosion on solid materials must also be considered since it can lead to the formation and expansion of cavities and grooves .
this in turn provides breeding sites for microorganisms , thereby compromising the efficacy of cleaning and disinfection procedures .
the surface characteristics of the microorganisms themselves and the various environmental conditions encountered in agri - food industries ( organic materials , ph , temperature , water activity , etc . ) also influence microbial attachment to inert surfaces [ 2 , 912 ] .
once they have adhered to inert surfaces , the microorganisms may exhibit a greater degree of resistance to the chemical or natural cleaning and disinfecting agents used in the agri - food industries compared to bacteria in suspension [ 13 , 14 ] .  
the potential for attachment and development of microorganisms on inert surfaces as well as the resistance of the resulting sessile cells has been and continues to be extensively studied [ 1519 ] . although an understanding of the parameters that govern the adhesion of these bacteria to solid surfaces could help developing new prevention procedures at the initial stages of microbial adsorption , there are still too many unknown factors concerning the adhesion capacity of the main food pathogens .   
the objective of this study was to find out the adhesion capacity of pathogens such as aeromonas hydrophila , escherichia coli o157:h7 , salmonella enteritidis , and staphylococcus aureus on two commonly used materials in food processing plants ( polystyrene and glass ) .
the influence of culture medium ph on the rate of adhesion by these agents at the interfaces was also simultaneously evaluated .
for this study , aeromonas hydrophila atcc 7966 , escherichia coli o157:h7 atcc 35150 , salmonella enteritidis e1347 , and staphylococcus aureus atcc 29213 were selected .
cryotubes of these strains , stored at 80c in tsb - ye ( tryptic soy broth supplemented with 1% yeast extract ; difco laboratories , detroit , mi ) containing 20% glycerol ( difco laboratories , detroit , mi ) , were thawed and the bacterial cultures were revived by two successive precultures in 10  ml of tsb - ye ( 1% v / v ) and then incubated for 24  h at 37c .
the harvested bacteria were washed three times and resuspended in buffers at ph 6.0 , 7.0 , or 8.0 .
a total viable count was performed for each culture and the total cfus determined using tryptic soy agar ( tsa ; difco laboratories , detroit , mi ) were between 4  10 and 2  10  cfu / ml .
polystyrene ( hydrophobic ) and glass ( hydrophilic ) substrates were selected for the adhesion tests .
25433 ; vwr international inc . , west chester , pa ) were used to obtain 5-cm coupons and 5-cm glass coupons were cut from microscope slides ( no . 48300 ; vwr international inc . ,
prior to physicochemical characterizations and adhesion tests , these substrates were soaked for 24  h in sodium hydroxide ( 1  n ) , washed and rinsed thoroughly eight times with deionized water ( millipore , billerica , ma ) .
the polystyrene coupons were sterilized for 5 min in boiling distilled water , while the glass coupons were directly autoclaved at 121c for 15  min in bioreactors .  
the attachment tests were conducted in sterile bioreactors ( bst model sc60 suspend reactor , biosurface technologies corporation , bozeman , mt ) . using a sterile clamp and under a microbiological hood ,
the sterile coupons were mounted on metal rods ( six rods per bioreactor ) in pairs , separated by a stainless steel nut . to ensure a sufficient attachment of the bacterial cells ,
the cultures of the pathogens in the bioreactors were left in contact with the inert surfaces for 24  h at ambient temperature ( 20  2c ) under low agitation ( 90  rpm ) . each experiment ( bacterium - culture ph - surface type combination ) was repeated three times and the means were used for the statistical analyses . to recover the sessile cells ,
two coupons of each material were removed from the rods using a sterile clamp and rinsed twice in tubes containing 10  ml of saline ( one tube per rinse , carefully rotating the tubes ) in order to eliminate the cells that had not adhered .  
the substrates were then placed in a tube containing 10  ml of sterile phosphate buffer ( oxoid ltd , basingstoke , hampshire , england ) and all of the adhered bacterial cells were detached in a sonication bath ( vwr international inc . , west
the tubes were vortexed for 30  s before the microbial counts were performed .  
after preparation of serial dilutions , the bacterial counts were determined by plating on tsa ( tryptic soy agar ; difco laboratories , detroit , mi ) incubated at 37c for 24  h. the relative adhesion ( % ) was estimated using the following formula : 
 ( 1)relative  adhesion  ( % )  = ( adhered  bacteria / initial  concentration)100 . 
  
in preliminary work , the two types of noncontaminated sterile materials were gold coated and observed under a scanning electron microscope in order to characterize the microstructure of the substrates .   
after two saline rinses , the contaminated coupons were fixed by immersion in 5  ml of 2.5% glutaraldehyde ( v / v ) in a 0.1  m sodium cacodylate buffer ( ph 7.3 ) and left at room temperature for 2  h. the glutaraldehyde was then removed using a pasteur pipette and the substrates were rinsed four times by immersing them for 15  min in 0.1  m sodium cacodylate buffer ( ph 7.3 ) .
dehydration was performed through an ascending series of ethanol ( approximately 5  ml ) concentrations ( 30% , 50% , 70% , and 80% ) for 15  min for each concentration , and then three times for 15  min in 100% ethanol .
dehydration was completed using co2 in a critical point dryer ( model e3000 cpd , bio - rad , polaron equipment ltd .
the samples were then mounted on an aluminum platform and covering with 8  nm of gold using a sputter coater ( cressington 108 , kurt j. lesker co. , clairton , pa ) .  
the substrates were observed under a scanning electron microscope ( hitachi s300n , hitachi , tokyo , japan ) . for the inert material , sterile dried substrates were positioned on a microscope stage for contact angle measurements .
a microsyringe ( chromatographic specialities inc . , brockville , ontario , canada ) was used to place a drop ( 1  l ) of each wetting agent  bidistilled water ( barnstead fistreem glassstill , england ) , formamide , and -bromonaphthalene ( aldrich chemical co. , inc . , milwaukee , wi)on the solid surfaces .  
the contact angles formed by these liquids were determined using a goniometer coupled with a 100  telescope ( gaertner scientific corp . ,
chicago , ill . ) .    in each case , six measurements were taken with each liquid on each substrate to determine the surface charge of the substrates   as well as their hydrophilic - hydrophobic characteristics .
the total surface energy (  ) of the solid substrates , the lifshitz - van der waals contribution to surface energies (  ) and the lewis acid - base bonds (  ) were calculated using an extension of the young - dupr equation : 
 ( 2)(1+cos  )lt=2[(slsllw)+(s+l )  + ( l+s)],tot=slw2(s+s)=slwsab , 
							 where  represents the angle formed by the wetting liquid on the substrate and l ,   the surface tension of the wetting liquid .  
( l , l , l ) and ( s , s , s ) are , respectively , the lifshitz - van der waals dispersion , electron donor ( basic ) and electron acceptor ( acid ) components of the wetting liquid and inert surface .
once these parameters have been determined , for both the solid substrates ( s ) and for each bacterium ( b ) in an aqueous medium ( l ) , the free energy of microbial adhesion , gadh(41 ) ,   was estimated according to the following formula : 
 ( 3)gadh=(slwblw)2(blwllw)2  (slwllw)2  + 2[l+(s+bl )  + b(s++b+l )  (s+b)(sb+ ) ] . 
							 in theory , the energy balance is favourable to bacterial adhesion if   gadh < 0 and unfavourable if gadh > 0
. bacteria physicochemical properties were determined for bacterial cultures grown at phs 6 , 7 , and 8 .
the contact angle measurements were performed on bacterial lawns deposited on filter paper as described by mafu et al .   and the total surface energy (  ) of the bacteria and the lifshitz - van der waals ( s ) contribution to surface
energies as well as the lewis acid - base bonds ( s ) were determined with the help of young - dupr relationship ( 2 ) .
the surface properties of the pathogens were evaluated in term of free energy of aggregation using the van der mei et al .  
approach  ( 4 ) 
 ( 4)gmwm=4(mlwwlw)2  4((m+m)+(w+w )  (m+w)(mw+ ) ) , 
							 where ( m , m , m ) and ( w , m , m ) are , respectively , the lifshitz - van der waals dispersion , electron donor ( base ) and electron acceptor ( acid ) components of the microorganisms studied and those of the water .
a preference for the aqueous medium , a characteristic of hydrophilic cell surfaces , is demonstrated by a gmwm value > 0 ,   while hydrophobic organisms , which tend to agglomerate in aqueous suspensions , are characterized by a gmwm < 0 .
the data were analysed using the glm procedure of sas software version 8.0 ( sas institute inc . , cary , nc ) .
an analysis of variance in the form of a factorial experiment ( bacterium  culture ph  surface type ) in complete blocks was chosen for adhesion tests .
when the ph - pathogen interaction terms were significant , a one - way analysis of variance ( culture ph ) was performed for each of the four bacteria .
the significant differences for the free energies of adhesion were detected using a one - way analysis of variance .
the substrates microstructure revealed that glass was a smooth surface ( figure 1(a ) ) , while polystyrene had irregularities , with tiny bumps and hollows ( figure 1(b ) ) .    the results obtained for the surface physicochemical properties of the two substrates , determined from contact angle measurement with three solvents , are detailed in table 1 .  
the surfaces were characterized by a similar lifshitz - van der waals (  ) dispersion component and by very low values for the electron acceptor parameter (  ) .
the electron donor (  ) and lewis acid base capacities (  ) showed considerable variability from one type of material to the other . the total surface energy (  ) measured for the polystyrene was lower than for glass .
water drops spread over the glass surface , demonstrating its hydrophilic character , consistent with the high value of the electron donor parameter .
in addition , the angles formed by the water on the specimen were very obtuse compared with those obtained on glass , enabling us to conclude that the surface is hydrophobic . the free energy of adhesion of the pathogens on bare surfaces , determined by combining the surface characteristics of the selected substrates ( table 1 ) and the surface properties of the bacteria ( table 4 ) , are shown in table 2 .
the gadh values of polystyrene were negative for all microorganism  culture ph  surface combinations , predicting that adhesion would be thermodynamically favourable for the polymer .
this approach predicts that conditions would be unfavourable to bacterial attachment in the presence of glass , a hydrophilic surface , since the free energy of adhesion was positive in all cases .  
theoretically , a. hydrophila and e. coli should exhibit weaker adsorption on both substrates at ph 6 ( p < .05 ) .   for s. aureus , the rate of adhered bacteria should be highest in the case of hydrophobic surface at ph 7 ( p < .05 ) and lowest in the case of hydrophilic surface at ph 8 ( p < .05 ) .
for the two substrates studied , ph of the culture affected the theoretical affinity of s. enteritidis ( p > .05 ) . at ph 6 , 7 , and 8
, no interactions were determined between ph of culture and adhesion capability for individual bacterium ( p > .05 ) .
the type of substrate did not influence the rate of attachment of pathogens ( p > .05 ) ,   however the effect of ph of the culture varied depending on the type of microorganism considered . 

e. coli displayed the lowest relative adhesion , regardless of environmental ph ( table 3 ) .
the affinity of e. coli for the solid samples was significantly influenced by ph ( p < .05 ) . 

a. hydrophila on solid surfaces exhibited the same profile as e. coli ( table 3 ) , however , aeromonas cell had greater affinity for the two substrates than the enterohemorrhagic bacterium , especially in acid and neutral media , with the number of attached cells ranging from 45% to 53%  ( p > .05 ) and from 39% to 42%  ( p > .05 ) ,   respectively .
the adhesion capacity of a. hydrophila to inert surfaces was lower after being cultivated in an alkaline medium ( p < .05 ) .
for e. coli and a. hydrophila , neutral ph was the best condition for attachment of these two pathogens , regardless of the type of surface .
type of substrate and ph had no effect on the adsorption of s. enteritidis to surfaces ( p > .05 ) .
the degree of contamination of polystyrene and glass by this enterobacteria was considerably higher than that observed for the other bacteria at ph 8 ( p < .05 ) . at ph 6 and 7 ,
s. enteritidis had been attached to the polymer and glass equally ( p > .05 ) . also , after the contamination period , adhesion of s. aureus to the two types of coupons ( table 3 ) was lower compared to the other experimental conditions ( p < .05 ) . the number of organisms attached to both substrates decreased by elevating ph . at ph 8 ,
the number of detached s. aureus cells was identical on both polystyrene and glass surfaces ( p > .05 ) . 

figure 2 demonstrates the mean relative adhesion rate of each bacterium as a function of the component of their surfaces .
in fact , since the solid surfaces had no effect on the concentration of adhered cells ( p > .05 ) ,   the mean between the relative adhesion to polystyrene and to glass could be calculated in order to obtain an overall adhesion rate . only an high correlation coefficient ( r = 0.94 ) between the free energy of aggregation of s. aureus and its mean rate of adhesion to the substrates has been found ( figure 2 ) .
the greater the hydrophilic character displayed by this bacterium , the lower its tendency to attach to inert surfaces . on both types of surfaces ,
porous and nonporous , a very low number of e. coli and dispersed cells of a. hydrophila were observed ( data not shown ) .
s. enteritidis cells were generally isolated or formed small clusters , regardless of the adhesion conditions ( figure 3 ) .
their dispersion was more uniform at ph 6 than at the other ph values ( figures 3(a ) and 3(b ) ) .
s. aureus invaded the abiotic surfaces in the form of single cells , small chains , or cell aggregates ( figure 4 ) .  
it was more difficult to observe staphylococci on polystyrene and glass at ph 8 ( figures 4(e ) and 4(f ) ) .  
as demonstrated by the present results , it becomes clear that in all case , no visible extracellular material was observed on both food contact surfaces .
bacterial adhesion to surfaces and biofilm formation are complex phenomena influenced by a number of factors .   in this study ,
three of these factors , namely , microbial strains , culture ph , and type of surface , were analysed .  
polystyrene and glass were chosen as surfaces , respectively , hydrophobic and hydrophilic , for the attachment and biofilm formation tests .   
scanning electron microscopy observation ( figures 3 and 4 ) showed that all the pathogens had the capacity to adhere to both types of surface .
although the cavities and distortions of the samples observed under the scanning electron microscope increase the specific contact area for bacteria , the differences in surface irregularities between both solid surfaces had no observable impact on the relative adhesion of the microorganisms .
these results are consistent with the findings of mafu et al . , who found no correlation between the microstructure of the materials and the capacity of bacteria to adhere to surfaces .  
also in this study , the attachment of solid surfaces by microorganisms was influenced solely by culture ph , which concurs with the work of husmark and ronner   as well as that of herald and zottola .  
the absence of fimbriae and curli [ 25 , 26 ] might explain the low contamination of e. coli observed in this study as well as in other experiments conducted on the contamination of polymers by serotype o157 [ 24 , 27 , 28 ] .  
the data from these experiments are not in agreement with the predictions deduced from the free energies of adhesion ( gadh ) of pathogens on polystyrene ( table 2 ) .
these energy characteristics demonstrated that conditions for adsorption of all microorganisms were thermodynamically favourable on polystyrene ( gadh < 0 ) and unfavourable on glass ( gadh > 0 ) surfaces .  
if the exposure time had been shorter ( maximum of four hours , which corresponds to irreversible bacterial adsorption ) , the predictions for the adhesion , based on gadh , might have proven valid .   in fact ,
the equation used to predict bacterial adhesion to surfaces ( gadh ) does not take into consideration the exopolysaccharides that might be produced for the irreversible attachment of microorganisms . in another attempt to explain adhesion detection with gadh values > 0 ,   we endeavoured to determine whether the surface properties of the infectious agents influenced their adhesion rate .  
thus , the free energy of aggregation of s. aureus could be correlated to the degree of attachment of the cocci ( figure 2 ) .  
gilbert et al .   also demonstrated that the adhesion of staphylococcus epidermidis to glass , unlike escherichia coli , was negatively correlated to the microorganism 's hydrophilicity and surface electronegativity .  
however , the physicochemical characteristics of the inert surfaces and of the other three pathogens examined throughout this study did not shed any light on their adhesion capacity .  
the explanation may lie in the composition of the suspension medium of the microorganisms , which was not taken into account in the estimation of ps and glass free energy . indeed ,
conditioning the substrates with compounds from the suspension medium can increase or decrease subsequent bacterial adhesion [ 23 , 30 , 31 ] .
these ionic substances located at the microorganism - surface interface can change ionic strength which is a critical factor in adhesion . some of these elements can reduce the repulsive force between the bacterium and the solid surfaces [ 32 , 33 ] , due to electrostatic interactions , by masking charges of the same sign .
thus , several stress responses are associated with the appearance of macromolecular agents in the medium .
many microorganisms exposed to acidic and alkaline stress synthesize polysaccharides , peptides , or heat - stable proteins [ 10 , 34 ] , which play an active role in induction of tolerance to ph stress .
for instance , synthesis by s. aureus of a surfactant , the toxin  ,   inhibits the action of d - alanine , limiting cell adhesion to polystyrene .
previous studies have also demonstrated the important roles played by cell organelles and bacterial mobility in transport and adhesion to various types of surfaces .  
pseudomonas fluorescens strains without flagella exhibited a weak capacity to attach to surfaces and develop a biofilm .
flagella apparently play an essential role during the initial reversible stages of attachment by overcoming the repulsive forces .
however , the nonmotile bacterium s. aureus adhered to the tested substrates at ph 6 in greater numbers than e. coli o157:h7 and a. hydrophila ( table 3 ) , which are motile microorganisms .
the presence of flagella is apparently more important when the velocity of the environmental liquid is high [ 4 , 36 ] .
mcclaine and ford   demonstrated that rotation of the flagellum caused detachment of fixed motile bacteria when flow rates were low ( 0.02 mlmin ) , while their adhesion was strengthened at high flow rates ( 2  mlmin  ) .   
although the irreversible attachment of microorganisms is often associated with the production of exopolysaccharides , no extracellular matrix was detected by scanning electron microscopy , regardless of experimental conditions .
this indicates that the presence of polymers is not necessary for permanent adhesion of microorganisms to a surface .
the visible filaments attached to the substrates may be dependent on the suspension medium .  
maximum polysaccharide production is generally associated with an environment deficient in essential nutrients , such as carbon , nitrogen , calcium , and iron [ 26 , 38 ] .
contact time and environmental temperature are other factors that influence the formation of a three - dimensional network [ 39 , 40 ]
. however , these appendices are crucial for biofilm consolidation and their resistance to environmental stresses .   
several molecules from the culture media can contribute to produce a conditioning film at the surface of the substrates .
these molecules include proteins , which are block copolymers presenting hydrophobic as well as hydrophilic sections .
it is thus possible for such molecules to adhere to hydrophilic substrates such as glass , resulting in an hydrophobization of the surface .
this phenomena would lead bacteria presenting the same adhesion trends on both ps and hydrophobically coated glass .
the results of this work indicate that pathogens could adapt to various ph levels of cultural media and adhere indifferently to inert polystyrene and glass surfaces , regardless of their hydrophilic or hydrophobic nature which leads to biofilm formation and increases the possibility of resistance to sanitizing agents . therefore , to minimize surfaces contaminations
, one should be aware of the influence of different environmental conditions which affects survival of bacteria in order to reduce their incidence in food systems .
the operations must be carried out promptly and regularly ( between two production cycles , for example ) , since they can effectively detach and eliminate the adhered bacteria when they have not yet formed a biofilm , thereby preventing contamination of raw materials and finished products and protecting consumer health .
 the adhesion of aeromonas hydrophila , escherichia coli o157:h7 , salmonella enteritidis , and staphylococcus aureus to hydrophobic and hydrophilic surfaces in cultures with different phs ( 6 , 7 , and 8) was studied . 
 the results indicated that the type of material had no effect on the attachment capacity of microorganisms , while environmental ph influenced the adhesion of a. hydrophila , e. coli , and s. aureus to both solid substrates . 
 the attachment of s. enteritidis ( p > .05 ) was not affected by the type of substrate or the culture ph , whereas e. coli displayed the weakest affinity for both polystyrene and glass surfaces . 
 no correlation was established between the physicochemical properties of the materials , or the bacterial and the rate of bacterial adhesion , except for s. aureus . 
 photomicrographs have shown that surfaces were contaminated by small clusters of s. enteritidis while s. aureus invaded the food contact surfaces in the form of small chains or cell aggregates . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion