pathogenic bacterial infections afflict countless numbers of individuals annually , with outcomes ranging from minor and treatable complications to death . food and waterassociated bacterial illnesses , estimated at 76 million illnesses per year , cost the us economy well in excess of $ 10 billion annually ( buzby and roberts , 1997 ; mead et  al . , 1999 ) .
development of accurate approaches for detecting and identifying diverse groups of pathogenic bacteria is essential for diagnosis and treatment of bacterial infections as well as assessing the toxicity potential of bacterial contamination . despite enormous advances in molecular and immunological ( dna and antibody ) detection technologies , pathogenic bacteria still escape detection .
detection methods based on protein or dna structure specific to an organism often can not distinguish between live and dead bacteria or the potential of these organisms to cause disease ( mckillip et  al .
, 1998 ; sheridan et  al . , 1998 ; nogva et  al . ,
previously , a cellbased biosensor consisting of cultured primary chromatophore pigment cells , specifically erythrophores , from betta splendens ( siamese fightingfish ) , was described in terms of its functionbased detection of toxic agents such as herbicides , fungicides , genotoxins ( mojovic et  al . , 2004 ) , cell receptor agonists and purified toxins ( dierksen et  al .
erythrophore cells are specialized pigmented nerve cells that can change appearance when physiologically stimulated ( elwing et  al . , 1990 ; karlsson et  al . , 1991 ; danosky and mcfadden , 1997 ) .
erythrophore cell response to toxic agents can be easily detected by monitoring changes in optical density due to movement of pigment organelles ( chromatosomes ) within the erythrophore cell .
pigment organelle movement can be dispersive ( pigment organelles move outwards toward the cell edge ) or aggregative ( movement towards the centre of the cell ) .
the movement of pigment organelles along a radically arrayed microtubule network achieves visual changes in erythrophore cell morphology .
pigment organelle movement is thought to be controlled through signal transduction pathways and other regulatory mechanisms in response to triggering events at various cell surface receptor sites , including gprotein coupled receptorbinding events ( danosky and mcfadden , 1997 ) .
the movement of the pigment organelles within erythrophore cells can be measured by recording the change in pigment area occupied by the erythrophore , allowing realtime assessment of erythrophore cell response to toxic conditions ( dierksen et  al . , 2004 ;
little is known about the potential of erythrophore cells to detect biologically active foodassociated bacterial pathogens .
bacillus cereus was selected as the initial model system because of its increasing implication in emetic and diarrheal forms of food poisoning ( turnbull , 1986 ; kramer and gilbert , 1989 ) .
in addition to its link to gastrointestinal infections , b. cereus , an opportunistic pathogen , has been linked to numerous nongastrointestinal infections including , osteomyelitis , pulmonary and wound infections , bacteremia and septicemia ( kramer and gilbert , 1989 ; turnbull , 1986 ) . in this study , we present findings in support of the potential for erythrophore cells to be used to detect , as well as potentially assess toxicity for , the foodassociated pathogenic bacteria , b. cereus , clostridium perfringens , clostridium botulinum and salmonella enteritidis .
a desirable feature of an erythrophore cellbased biosensor would be the ability to distinguish uninoculated bacterial culture medium ( representing the negative control ) from the cultured bacterial strains , thus erythrophore cell response to culture medium was included in this analysis .
cultured in bhi ( brain heart infusion ) medium : bacillus subtilis 1a1 ( a laboratory reference strain ; yoshida et  al . , 1995 ) , b. cereus atcc 49064 ( identified in a gastroenteritis outbreak and produces diarrheogenic enterotoxin ; midura et  al . , 1970 ; taylor and gilbert , 1975 ; melling et  al . , 1976 ) ,
b. cereus atcc 14579 ( frankland and frankland , 1887 ) and a b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 ( salamitou et  al . , 2000 ) . the negative control ( uninoculated bhi media ) caused slight dispersion of pigment organelles within the erythrophore cell ( fig . 
erythrophore cell response to b. subtilis 1a1 culture was similar in strength and rate to the response elicited by the negative control ( fig . 
1b and d ) , and this response was found to be statistically insignificant ( p  =  0.635 ) .
1c ) or atcc 14579 induced erythrophore pigment organelles to rapidly aggregate ; this response was detected within two to 5  min after addition of the bacterial culture ( fig . 
erythrophore cell response to b. cereus atcc 49064 or atcc 14579 was found to be statistically significant compared with the negative control ( p  <  0.001 and p  <  0.001 respectively ) .
erythrophore cells appearance at time 0 ( left ) and time 20  min ( right ) upon exposure to : ( a ) bhi medium , ( b ) b. subtilis 1a1 , ( c ) b. cereus atcc 49064 .
images are at 100 , the size bar represents a length of 100  m .
( d ) graphical display of erythrophore cell pigment area change in response to bacillus spp . a negative change in pigment area is indicative of pigment aggregation in erythrophore cells whereas a positive change in pigment area represents pigment dispersion .
(  ) bhi medium ; (  ) b. subtilis 1a1 ; (  ) b. cereus atcc 49064 ; (  ) b. cereus atcc 14579 ; (  ) b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 .
data represent the mean values of three trials . to investigate the potential mechanism of erythrophore cell response to b. cereus
, erythrophore cells were tested against a b. cereus atcc 14579 strain lacking the global gene regulator , plcr ( salamitou et  al . , 2000 ) .
the b. cereus plcr deletion mutant strain induced a similar response ( slightly dispersive rather than aggregative ) in erythrophore cells as the negative control ( bhi ) ( p  =  0.0754 ; fig . 
to further evaluate erythrophore cell response , we investigated the response of erythrophore cells to additional foodassociated pathogenic bacteria including , the gramnegative bacterium , s. enteritidis atcc 4931 and the grampositive spore forming bacterium , c. perfringens sm101 ( zhao and melville , 1998 ) . erythrophore cells were exposed to s. enteritidis atcc 4931 grown in luria  bertani broth ( lb ; fig . 
the difference between s. enteritidis and the negative control ( lb media ) was statistically significant ( p  <  0.001 ) .
clostridium perfringens sm101 was cultured in duncanstrong sporulation medium to allow production of the potent enterotoxin cpe ( elwing et  al .
clostridium perfringens induced aggregation of erythrophore pigment within 4  min followed by slow dispersion for the remainder of the assay ( fig . 
this response was very different from the dispersive response of the negative control ( duncanstrong sporulation medium ) and was statistically significant ( p  <  0.004 ) .
a negative change in pigment area is indicative of pigment aggregation whereas a positive change in pigment area represents pigment dispersion . 
a. (  ) lb medium ; (  ) s. enteritidis atcc 4931 . 
b. (  ) duncanstrong sporulation medium ; (  ) c. perfringens sm101 . 
clostridium botulinum was chosen as a second representative of a grampositive , spore forming , yet anaerobic bacterium associated with food contamination and potential use as a biological weapon .
erythrophore cells were challenged with c. botulinum nctc 7272 ( type a ) or c. botulinum nctc 7273 ( type b ) cultured in bhi .
erythrophore cell response was monitored for 1  h. very little response to c. botulinum was observed during this hour ; therefore monitoring time was extended to 6  h. two hours after exposure to c. botulinum nctc 7272 or nctc 7273 , pigment organelles began to slowly aggregate ( fig . 
3a ) and aggregation of pigment organelles was complete at approximately 5  h. the erythrophore response to both c. botulinum cultures was statistically significant compared with the negative control ( bhi ) ( c. botulinum nctc 7272 p  <  0.005 and c. botulinum nctc 7273 p  <  0.006 respectively ) . the observed morphological changes of the erythrophore cells were unique from changes induced by other bacterial pathogens . extensive dendrite formation was observed in erythrophore cells treated with c. botulinum ( fig . 
3c ) , while other bacterial pathogens induced pigment organelles to centrally localize within the erythrophore cell ( fig . 
( a ) b. splendens erythrophore cell response after 6  h exposure to bacterial isolate .
a negative change in pigment area is indicative of intracellular pigment aggregation whereas a positive change in pigment area represents pigment dispersion .
(  ) c. botulinum nctc 7272 ; (  ) c. botulinum nctc 7273 ; (  ) bhi medium .
( b ) and ( c ) b. splendens erythrophore cells at 100 , the size bar represents a length of 100  m ; left , time  =  0  h , right , time  =  6  h , exposure to : ( b ) bhi medium , ( c ) c. botulinum nctc 7272 .
this study demonstrates the potential of a novel bacterial biosensor populated with b. splendens erythrophore cells .
when erythrophore cells are exposed to a bacterial pathogen , the response is easily observed and quantified .
erythrophore cells respond to selected foodassociated bacteria , b. cereus , s. enteritis , c. perfringens and c. botulinum , and this response is different from the respective culture medium ( negative control ) .
erythrophore cells were unable to distinguish the nonpathogenic bacterial strains , b. subtilis and the b. cereus plcr mutant strain from the negative control .
the statistical and graphical evidence suggests that erythrophore cell response has potential for use as a biosensor in the detection or toxicity assessment for foodassociated pathogenic bacteria . what is it about a bacterial pathogen , such as b. cereus , that causes erythrophore cells to respond by relocating pigment organelles to the centre of the erythrophore cell ?
the failure of erythrophore cells to respond to the nonpathogenic b. cereus plcr mutant strain provides insight to this intriguing question .
plcr , a pleiotropic transcriptional regulator , controls the expression of numerous extracellular compounds , including several virulence factors ( agaisse et  al . , 1999 ;
the observation that b. cereus lacking plcr does not induce pigment aggregation in erythrophore cells suggests that failure to express one or several of the plcr regulated genes results in the failure of b. cereus to induce a response in erythrophore cells .
lereclus and coworkers have shown that b. cereus pathogenicity is dependent on plcr ( lereclus et  al . , 2000 ;
salamitou et  al . , 2000 ) , suggesting that erythrophore cells may interact with bacterial virulence traits or other gene products dependent on plcr .
identification of b. cereus mutants that no longer induce a response in erythrophore cells is currently underway .
identifying the mutant bacterial gene products will define the mechanism(s ) by which this bacterial pathogen induces a change in pigment organelle location within erythrophore cells .
sm101 cause a slower rate of aggregation and less overall aggregation when compared with b. cereus atcc 49064 and atcc 14579 , suggesting that bacterial pathogens may interact with erythrophore cells through different mechanisms .
additionally , when erythrophore cells are exposed to c. botulinum , the erythrophore cell morphology in response to this bacterium is different from reactions with other bacterial pathogens .
the observation of specific erythrophore cell morphology in response to specific bacterial pathogens as well as differences in the kinetics of erythrophore cell response may provide another means to measure and characterize bacterial detection .
reports of salmonella contaminated tomatoes , peanut butter and spinach and c. botulinum contaminated canned meats indicates the ease by which foodassociated bacterial pathogens evade detection , often resulting in massive recalls of popular food items .
given the economic impact of massive recalls , as well as the lack of effective detection methods to enforce the usa 's and uk 's zero tolerance policy for certain foodassociated pathogenic bacteria , suggests a need for detection methods based on parameters different from those describing bacterial presence .
although newer detection methods can identify single microbes , thus meeting zero tolerance requirement , many technical challenges remain for these methods .
these challenges often include time intensive sampling and testing practices , long culture times to increase the number of bacteria to detectable levels , and costly shipment methods to move samples to a central laboratory for analysis ( cady et  al . , 2005 ; batt , 2007 ) .
furthermore , many of these methods rely on detecting a structure through the use of a dna or antibody probe , and thus are limited in the evaluation of toxicity and the potential of bacteria to cause disease .
additionally , dna or antibodybased detection methods are limited in the detection of unidentified ( e.g. genetically rearranged ) or newly emerging ( e.g. new toxin producing variants ) pathogenic bacteria .
while dna technology such as pcr may be able identify offending bacteria in the food , it can not assess if the detected bacteria are alive and exhibiting the pathogenic behaviour that make them a health risk .
for example , the bacteria may be nonviable and not exhibiting pathogenic behaviour , but their dna may still be detectable by pcr technology , thus suggesting that information gathered from dna characterizing techniques , if taken alone , may be seriously misleading . the b. splendens erythrophore cell response described here represents a unique class of cellbased biosensors with potential to complement current detection methods as well as circumvent a number of limitations and challenges with the available technology .
all bacillus spp . [ b. subtilis 1a1 ( yoshida et  al . , 1995 ) ,
b. cereus atcc 14579 ( frankland and frankland , 1887 ) and a b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 ( salamitou et  al . , 2000 )
clostridium perfringens sm101 ( zhao and melville , 1998 ) was generously provided by dr mahfuzur r. sarker .
briefly , a 0.1  ml aliquot of a frozen stock of c. perfringens sm101 was transferred into 6  ml of fluid thioglycollate ( ftg ) and then heat shocked for 20  min at 70c .
the heatshocked culture was then incubated for 14  h at 37c , and 0.4  ml of this starter culture was transferred to a second 6  ml ftg before this culture was incubated for 9  h at 37c ( kokaikun et  al . , 1994 ) .
an aliquot ( 0.4  ml ) of this ftg culture was then added to 20  ml of duncan strong ( ds ) sporulation medium ( mcdonel et  al . ,
1988 ) and incubated for 8  h at 37c ( mcdonel et  al . , 1988 ) .
clostridium botulinum nctc 7272 and c. botulinum nctc 7273 were obtained from the national collection of type cultures , phls central public health laboratory . a 0.1  ml aliquot of a cooked meat medium stock of c. botulinum nctc 7272 and nctc 7273 was transferred to 1.0  ml of bhi medium .
a 0.1  ml aliquot of the starter culture was transferred to a second 1  ml bhi tube before the culture was incubated for 24  h at 37c .
all c. botulinum culturing was done in an anaerobic chamber using anaerogen gas packs ( fisher ) .
all biosafety level 2 ( bsl2 ) bacterial agents were cultured in an approved bsl2 facility and performed under the requirements and regulations of and approved by oregon state university 's institutional biosafety committee .
erythrophore cells were isolated from the tails and fins of b. splendens fish as described previously ( mojovic et  al . , 2004 ) .
briefly , tissue was washed in skinning solution [ 1  mm naedta , 5.6  mm glucose and penicillin 
streptomycin mixture 1:100 ( w / v ) in calcium and magnesiumfree phosphatebuffered saline ( pbs ) ] , digestion with an enzyme solution [ 2030  mg of collagenase type 1 ( worthington ) , and 13  mg of hyaluronidase ( worthington ) , in 7  ml of pbs ] .
after several washes , the cell pellet was resupsended in a predetermined volume of leibovitz l15 medium+ ( l15 + , gibco # 21083027 , 500  ml of dyefree l15 was supplemented with 10  ml 1  m hepes ( gibcobrl 15630080 ) buffer and 5  ml antibioticantimycotic ( gibcobrl 15240062 ) to ensure the desired plate density in the centre of each well of a 24well microtitre plate or 48well microtitre plate .
the l15 + medium was added in each well to a final volume of 1.5  ml ( 24well microtitre plate ) or 0.75  ml ( 48well microtitre plate ) and 5% fetal bovine serum ( hyclone # sh3007101 ) was added to each well .
validation tests were performed for each preparation of erythrophore cells to ensure that isolated erythrophore cells were physiologically responsive .
erythrophore cells were treated with a known dispersive agent , melanocyte stimulating hormone ( msh ) , and a known aggregative agent , clonidine , to verify physiological responsiveness ( dierksen et  al . , 2004 ; mojovic et  al . , 2004 ) .
this effort was performed under the requirements and regulations of and approved by oregon state university 's institutional animal care and use committee , approval # 2979 and # 3513 .
monitoring erythrophore cell response is based on observing and recording changes in the pigmented area of erythrophore cells exposed to bacterial agents and control agents as previously reported ( dierksen et  al .
a field of view containing approximately 100 erythrophore cells was monitored under a magnification of 100 .
bacterial suspension or physiological response testers ( 400  nm clonidine , sigma c7897 ; 400  nm msh , sigma m4135 , respectively ) was added to each well to a final dilution of 1:10 in l15 medium ( viable cell counts were 10 for bacillus spp . and 10 cells salmonella and clostridium spp .
respectively ) . digital images ( jpeg format ) at 300    300 resolution were taken using a spot insight 320 colour camera ( diagnostic instruments , sterling heights , mi , usa ) and spot software version 3.5.6.2 ( diagnostic instruments ) , through a leica ( leica , wetzlar , germany ) dmil inverted microscope ( bartels and stout ) .
erythrophore response was recorded for 20  min ; images were captured at 0 , 0.25 , 0.5 , 1 , 2 , 4 , 10 and 20  min . for experiments lasting 6  h ,
images were captured at the same time interval as the 20  min experiments , then every 10  min up to 60  min .
after 60  min , images were captured every 30  min until the experiment was completed .
the images were opened in image pro plus 4.1 ( media cybernetics ) , exported and processed into excel .
the total area in pixels ( digital area units ) for each captured image was determined and the percentage pigmented area change [ cell area change ( % )  =  (a0    ax)/a0    100 , where a0 is the initial area and ax is the final area ] .
an aggregative response would result in a negative per cent area change and a dispersive response would result in a positive per cent area change .
the statistical analysis was carried out with s plus statistical software ( insightful technologies , seattle , wa ) .
all bacterial challenges were completed in triplicate using three different erythrophore cell preparations ; reported values are the average of three trials .
final time points were analyzed using a twosample ttest , all reported pvalues were declared significant at p  <  0.05 .
all bacillus spp . [ b. subtilis 1a1 ( yoshida et  al . , 1995 ) ,
b. cereus atcc 14579 ( frankland and frankland , 1887 ) and a b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 ( salamitou et  al . , 2000 )
clostridium perfringens sm101 ( zhao and melville , 1998 ) was generously provided by dr mahfuzur r. sarker .
briefly , a 0.1  ml aliquot of a frozen stock of c. perfringens sm101 was transferred into 6  ml of fluid thioglycollate ( ftg ) and then heat shocked for 20  min at 70c .
the heatshocked culture was then incubated for 14  h at 37c , and 0.4  ml of this starter culture was transferred to a second 6  ml ftg before this culture was incubated for 9  h at 37c ( kokaikun et  al . , 1994 ) .
an aliquot ( 0.4  ml ) of this ftg culture was then added to 20  ml of duncan strong ( ds ) sporulation medium ( mcdonel et  al . ,
1988 ) and incubated for 8  h at 37c ( mcdonel et  al . , 1988 ) .
clostridium botulinum nctc 7272 and c. botulinum nctc 7273 were obtained from the national collection of type cultures , phls central public health laboratory . a 0.1  ml aliquot of a cooked meat medium stock of c. botulinum nctc 7272 and nctc 7273 was transferred to 1.0  ml of bhi medium .
a 0.1  ml aliquot of the starter culture was transferred to a second 1  ml bhi tube before the culture was incubated for 24  h at 37c .
all c. botulinum culturing was done in an anaerobic chamber using anaerogen gas packs ( fisher ) .
all biosafety level 2 ( bsl2 ) bacterial agents were cultured in an approved bsl2 facility and performed under the requirements and regulations of and approved by oregon state university 's institutional biosafety committee .
erythrophore cells were isolated from the tails and fins of b. splendens fish as described previously ( mojovic et  al . , 2004 ) .
briefly , tissue was washed in skinning solution [ 1  mm naedta , 5.6  mm glucose and penicillin 
streptomycin mixture 1:100 ( w / v ) in calcium and magnesiumfree phosphatebuffered saline ( pbs ) ] , digestion with an enzyme solution [ 2030  mg of collagenase type 1 ( worthington ) , and 13  mg of hyaluronidase ( worthington ) , in 7  ml of pbs ] .
after several washes , the cell pellet was resupsended in a predetermined volume of leibovitz l15 medium+ ( l15 + , gibco # 21083027 , 500  ml of dyefree l15 was supplemented with 10  ml 1  m hepes ( gibcobrl 15630080 ) buffer and 5  ml antibioticantimycotic ( gibcobrl 15240062 ) to ensure the desired plate density in the centre of each well of a 24well microtitre plate or 48well microtitre plate . the l15 + medium was added in each well to a final volume of 1.5  ml ( 24well microtitre plate ) or 0.75  ml ( 48well microtitre plate ) and 5% fetal bovine serum ( hyclone # sh3007101 ) was added to each well .
validation tests were performed for each preparation of erythrophore cells to ensure that isolated erythrophore cells were physiologically responsive .
erythrophore cells were treated with a known dispersive agent , melanocyte stimulating hormone ( msh ) , and a known aggregative agent , clonidine , to verify physiological responsiveness ( dierksen et  al . , 2004 ; mojovic et  al . , 2004 ) .
this effort was performed under the requirements and regulations of and approved by oregon state university 's institutional animal care and use committee , approval # 2979 and # 3513 .
monitoring erythrophore cell response is based on observing and recording changes in the pigmented area of erythrophore cells exposed to bacterial agents and control agents as previously reported ( dierksen et  al .
a field of view containing approximately 100 erythrophore cells was monitored under a magnification of 100 .
bacterial suspension or physiological response testers ( 400  nm clonidine , sigma c7897 ; 400  nm msh , sigma m4135 , respectively ) was added to each well to a final dilution of 1:10 in l15 medium ( viable cell counts were 10 for bacillus spp . and 10 cells salmonella and clostridium spp . respectively ) .
digital images ( jpeg format ) at 300    300 resolution were taken using a spot insight 320 colour camera ( diagnostic instruments , sterling heights , mi , usa ) and spot software version 3.5.6.2 ( diagnostic instruments ) , through a leica ( leica , wetzlar , germany ) dmil inverted microscope ( bartels and stout ) .
erythrophore response was recorded for 20  min ; images were captured at 0 , 0.25 , 0.5 , 1 , 2 , 4 , 10 and 20  min . for experiments lasting 6  h ,
images were captured at the same time interval as the 20  min experiments , then every 10  min up to 60  min .
after 60  min , images were captured every 30  min until the experiment was completed .
the images were opened in image pro plus 4.1 ( media cybernetics ) , exported and processed into excel . the total area in pixels ( digital area units ) for each captured image was determined and the percentage pigmented area change [ cell area change ( % )  =  (a0    ax)/a0    100 , where a0 is the initial area and ax is the final area ] .
an aggregative response would result in a negative per cent area change and a dispersive response would result in a positive per cent area change .
the statistical analysis was carried out with s plus statistical software ( insightful technologies , seattle , wa ) .
all bacterial challenges were completed in triplicate using three different erythrophore cell preparations ; reported values are the average of three trials .
final time points were analyzed using a twosample ttest , all reported pvalues were declared significant at p  <  0.05 .
 summarycellbased biosensors have been proposed for use as functionbased detectors of toxic agents . 
 we report the use of betta splendens chromatophore cells , specifically erythrophore cells , for detection of foodassociated pathogenic bacteria . 
 evaluation of erythrophore cell response , using bacillus spp . 
 , has revealed that this response can distinguish pathogenic bacillus cereus from a nonpathogenic b. cereus plcr deletion mutant and a nonpathogenic bacillus subtilis . 
 erythrophore cells were exposed to salmonella enteritidis , clostridium perfringens and clostridium botulinum . 
 each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium , and this observed response was unique for each bacterial pathogen . 
 these findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for foodassociated pathogenic bacteria . 
Introduction
Results
Discussion
Experimental procedures
Bacterial culture preparation
Erythrophore cell preparation
Erythrophore cell response and computer analysis
Statistical analysis