shiga - toxin- ( stx- ) producing escherichia coli strains , including e. coli o157:h7 , are a worldwide cause 
 of human disease with a wide spectrum of symptoms ranging from mild diarrhea to 
 life - threatening hemolytic - uremic syndrome ( hus ) . the stx - producing family of 
 human disease - associated e. coli is 
 also characterized by its diversity of toxin type ( i.e. , lysogenic either for 
 the stx1-encoding phage or the stx2-encoding phage , or with both lysogens ) and 
 by its o : h serotype range [ 1 , 2 ] . in spite of 
 diverse virulence characteristics , one common trait that emerges very clearly 
 is that most of these strains have the ability to withstand gastric acidity [ 36 ] or the conditions in acidic foods [ 79 ] . it is noteworthy that acid tolerance plays a vital role in the survival and 
 virulence of diarrheagenic e. coli strains [ 1012 ] . the ability of e. coli o157:h7 strains to survive in 
 acidic conditions has been studied extensively [ 1316 ] , but there are few reports about 
 the tolerance of non - o157 stec serogroups to organic acids in foods [ 17 , 18 ] . it is thought that acid resistance 
 and/or induction of acid tolerance may enable pathogens to survive 
 gastrointestinal acidity better and so , ultimately , cause disease [ 1922 ] . cattle 
 are considered to be a major reservoir of e. 
 coli o157:h7 for human infection . the pathogen has been isolated 
 from raw milk , and multiple outbreaks of e. coli o157:h7 linked to the ingestion 
 of raw milk and dairy products have been reported [ 25 , 26 ] . several authors have studied the 
 ability of e. coli o157:h7 to grow 
 and survive in different types of cheese . in fresh cheese , e. coli o157:h7 increased by 2 log ( cfu per gram ) during cheese 
 manufacture , but total inactivation was 
 obtained during heat treatment ( 57c for more than 1.5 hours ) of the curd and whey . reitsma and henning ( 1996 ) found that this microorganism was able to grow 
 during cheddar cheese manufacture , even with an initial inoculum in milk of 
 only 1 cfu ml . e. coli o157:h7 also survived the manufacture and storage of camembert and feta cheeses 
 at 2  1c for 65 and 75 days , respectively . moreover , this pathogen was able to 
 survive all stages of smear - ripened cheese production for up to 70 days 
 postmanufacture . e. coli o157:h7 was not eliminated from goat 's milk lactic cheese , made with raw milk , 
 because this organism tolerates the low ph   and low temperatures [ 31 , 32 ] which characterize the 
 manufacturing and ripening process . surprisingly , studies on contamination 
 of milk , or its products , with non - o157 e. coli have been limited . two studies from france 
  have reported stec 
 prevalence in cheeses [ 33 , 34 ] , 3 studies reported results for 
 ewes ' and caprine milk cheeses [ 3537 ] and , very recently , a study 
 described the prevalence of stec in swiss raw milk cheeses . moreover , there are no published 
 experiments evaluating the growth or survival of non - o157 stec in cheeses . however , non - o157 e. coli infections are , within european 
 member states , considered to be at least as important as e. coli o157:h7 infections though they are thought to be generally 
 underdiagnosed . for example , in italy , denmark , and germany , more than 40% of the confirmed 
 cases of stec - related hus were caused by non - o157 stec . more recently , in 2005 , 
 french raw milk camembert - type cheeses contaminated by escherichia coli o26 and o80 caused 16 hus cases and a national and 
 international recall of the entire production of cheeses . other human infections with 
 non - o157 stec from dairy products are 
 well documented [ 4447 ] . these outbreaks suggest that the 
 acid tolerance of the non - o157 stec strains , like the o157 stec strains , 
 enables these bacteria to survive in moderately acidic food . acid tolerance is 
 defined by the growth of log - phase cultures at a moderately low ph ( ph 5.5 to 
 6.0 ) inducing mechanisms of survival in the more extreme acid conditions of ph 
 2.5 . a similar phenomenon , termed the 
  acid tolerance response , the purpose 
 of the present work was to address the question of whether the acidic 
 resistance confers an ecological superiority . this potential ecological 
 superiority of stec strains was evaluated by the ability of acid - resistant ( ar ) 
 and non - acid - resistant ( nar ) stec strains to survive the fermentation process of 
 camembert - type cheeses . in a previous study , we have 
 investigated the fate of e. coli o157:h7 during the manufacture and ripening of raw goat 's milk lactic cheeses 
 and noted that e. coli o157:h7 was 
 able to survive the cheese - making process . the main objectives of the present 
 study were firstly to evaluate the growth and survival of ar and nar shiga - toxin - producing e. coli strains and , secondly , to 
 check whether microfiltered milk camembert cheeses could induce acid tolerance 
 in inoculated nar stec strains . a collection of 62 stec strains were 
 isolated during previous french epidemiological studies , whose purpose was to 
 determine stec contamination prevalence in dairy products , in pork , and in the 
 environment [ 34 , 5254 ] . with the aim of evaluating the 
 ability of these bacteria to survive exposure to acid these mechanisms are as follows : ar1 : oxidative 
 system ( glucose  repressed system),ar2 : system 
 depending on the presence of glutamate , ar3 : system 
 depending on the presence of arginine . it was tested according to the method previously 
 described by large et al . . ar1 : oxidative 
 system ( glucose  repressed system ) , ar2 : system 
 depending on the presence of glutamate , ar3 : system 
 depending on the presence of arginine . four ar 
 stec and four nar stec strains were selected from a previous study ( data not 
 shown ) . the survival rate of the strains in the presence of glucose and amino 
 acids is displayed in table 1 . two 
 spontaneous nalidixic acid - resistant mutants were selected in vitro for each ar and nar stec 
 strain , previously selected by plating stec on bhi agar containing nalidixic 
 acid ( 0.1 to 10 g / ml ) in increasing concentrations , using the protocol 
 described by truong et al . . the antibiotic 
 susceptibility of these ar or nar mutants is shown in table 1 . to follow the growth of these strains during the 
 manufacture and ripening of the camembert cheese , spontaneous 
 antibiotic - resistant derivatives ( nalidixic acid , rifampicin , and 
 spectinomycin ) we carefully checked that the chosen resistant 
 derivatives were neither affected in their growth rate nor in their 
 acid - resistance properties ( data not shown ) . raw cow milk samples were collected 
 aseptically from the bulk storage tank after it had been cooled to < 5c 
 and maintained refrigerated at 3c for transportation to actilait . raw milk was 
 microfiltered at 40c through a 1.4 m ceramic membralox membrane ( pall exekia , bazet , france ) . the microfiltered cow milk was 
 analyzed , in order to check that it was not contaminated by stec , by performing 
 a polymerase chain reaction ( pcr ) for shiga - toxin coding genes ( stx genes ) after an enrichment step , as 
 described by fremaux et al . . cultures of 
 antibiotic - resistant stec strains were maintained at   80c in 
 cryopreservative beads ( starlab , bagneux , 
  france ) . prior 
 to preparing the inoculation mixtures , each culture of antibiotic - resistant 
 stec strains was incubated overnight ( 18 hours ) in lb broth at 37c . strains were grown 
 individually at 37c for 24 hours in lb broth , with or without antibiotics , to reaffirm that the 
 strains retained resistance . cells were washed twice in 0.1% peptone water and suspended 
 in 0.1% peptone water to achieve the required inoculation ( 10 cfu ml ) level by standardization of the absorbance at a600 nm using a spectrophotometer ( biophotometer , eppendorf , le pecq , france ) . they 
 were designed corresponding with the need to have 3 different antibiotic - resistant 
 or non - resistant phenotypes in the same mixture . the 6 mixtures of stec strains 
 used for this experimentation are detailed in table 2 . the stec population in each mixture 
 was confirmed by plating 100 l of the suspension onto lb - a plates supplemented 
 with the appropriate antibiotic to verify the initial inoculum levels . six 
 millilitres of mixture were inoculated into 6.5 l of milk to obtain a final 
 concentration in the microfiltered milk of approximately 10 cfu ml of stec . twelve batches of cheeses were 
 prepared ( two batches per mixture ) and 4 batches of uninoculated cheese samples 
 served as negative controls ( 1 batch per manufacturing day ) . artisanal microfiltered cow 's milk 
 lactic cheeses were prepared following the industrial specifications of the 
 french institute of cheese ( actilait ) . the milk 
 was inoculated ( 2  10 cfu per 100 kg ) with mesophilic 
 starters mm100 ( lactococcus lactis subsp . lactis , cremoris , and lactis biovar diacetylactis ) , ( rhodia , 
 dang - saint - romain , france ) as well as ripening flora penicillium camemberti ( pc p9 , 4 doses per 1000 kg cargill , st germain 
 en laye , france ) , geotrichum candidum ( gca , 2 doses per 1000 kg , 
 cargill ) and 10.5 ml of a solution of cacl2 was also added per 100 kg 
 of milk . the 
 inoculated milk was matured for 2 hours at 32c and renneting was 
 carried out using 30 ml of rennet per 100 l of 
 milk ( at 530 mgl of 
 chymosin , berthelot , abia s.a . the 
 milk coagulated after 8 minutes and was left undisturbed until the ph decreased 
 to 6.15 . the curd was cut into 3 cm cubes , healed for 
 30 minutes ( ph minimum = 6.00 ) , 
 drained and transferred to 77  110 mm cylindrical plastic moulds . cheeses weighing approximately 250 g were removed from the 
 mould , and the temperature of the cheese - making chamber was reduced to 2022c . after 20 hours , the 
 cheeses were plunged into a saturated brine solution at 10c for 25 minutes . cheeses 
 were dried at 13c for 5 hours and matured at 11c and 95% relative humidity for 20 days , then finally packaged and stored at 4c . the manufacturing protocol for lactic cheeses 
 made with microfiltered is outlined in figure 1 . physicochemical and bacteriological 
 analyses were performed at the following steps.step 1 : microfiltered 
 milk before the maturation 
 stage.step 2 : curd during 
 draining ( 3 hours 20 minutes).step 3:cheese at the 
 end of the moulding stage ( 3 hours 45 minutes).step 4:cheese after 
 salting ( 1 day).step 5:cheese after 
 drying ( 1 day).step 6:cheese at the 
 middle of the ripening stage ( 10 days).step 7:cheese at the 
 end of ripening ( 20 days ) .   microfiltered 
 milk before the maturation 
 stage cheese at the 
 end of the moulding stage ( 3 hours 45 minutes ) . cheese at the 
 end of ripening ( 20 days ) . during 
 cheese manufacture , 25 ml samples of microfiltered milk or 
 25 g of drained curd or cheese 
 ( rind and core ) were sampled and homogenized with 225 ml of buffered peptone 
 water ( bpw , biomrieux , marcy l'etoile , france ) in a sterile bag filter 
 ( bagsystem 400 ml model+ , interscience , saint nom la breteche , france ) and 
 stomached for 30 seconds ( stomacher mix1 , aes laboratory , bruz , france ) . the filtered liquid was diluted in bpw and spread , using spiral plating ( wasp 
 spiral plating , aes laboratory , bruz , france ) , onto lb - a plates . lb - a plates 
 were supplemented with rifampicin ( 100 g ml ) , spectinomycin ( 100 g ml ) , or nalidixic acid ( 40 g ml ) , when 
 appropriate , and incubated for 24 hours at 37c . enumeration of the 
 colonies was performed with an automatic colony counter ec2 easy count 2 ( aes 
 laboratory , bruz , france ) . the ph was measured for each cheese 
 ( inoculated or not inoculated ) at each time of sampling . for the chemical 
 measurements , analyses were performed on the raw milk after microfiltration and 
 on the noninoculated ( negative controls ) cheeses after 1 day ( before brining ) 
 and 20 days ( end of the ripening stage ) . milk fat 
 content was determined by the acido - butyrometric 
 method of gerber , according to afnor 
 ( nf v 04 - 210 ) , and the milk protein rate was obtained using the amido black 
 method ( afnor , nf 
  v 04 - 216 ) . cheese moisture content was determined with an infrared - dryer 
 ( prcisa xm60 ) , according to afnor ( nf v 04 - 282 ) , and the cheese fat content 
 was measured by the heiss butyrometric method 
 . the ripened cheese salt content was determined using a chloride 
 analyzer ( afnor , nf v 04 - 288 ) . cheese phs were measured using a 
 penetration electrode ( ph meter 330 , fisher bioblock scientific , f67403 
 illkirch cedex , france ) . simulated gastric fluid was prepared accorded to the 
 protocol used by yuk and marshall 
 . g l of proteose - peptone ( fluka - biochemika , switzerland ) , 3.5 g l of 
 d - glucose ( fluka - biochemika , switzerland ) , 2.05 g l of 
 nacl ( sigma - aldrich , lyon , france ) , 0.6 g l of kh2po4 ( merck sharp 
 & dohme , paris , france ) , 0.11 g l of cacl2 ( sigma - aldrich , lyon , france ) , 
 0.37 g l of kcl ( sigma - aldrich , lyon , france ) , 0.05 g l of ox 
 bile ( sigma - aldrich , lyon , france ) , 0.1 g l of lysozyme 
 ( sigma - aldrich , lyon , france ) , and 13.3 mg l of pepsin 
 ( sigma - aldrich , lyon , france ) . the final ph was adjusted to 1.5 using sterile 
 5.0 n hcl ( merck sharp & dohme , paris , 
  france ) . all 
 compounds were autoclaved separately , except for the ox bile , lysozyme , and 
 pepsin which were filter sterilized , followed by aseptic mixing . however , 90 ml of sgf ( ph 1.5 ) , at 37c , were added aseptically 
 to 10 g of inoculated cheese sampled at the end of the ripening stage . to obtain a 
 final inoculated sgf at ph 2.5 , the ph of each sample was lowered to 2.5 using 
 ( 5.0 n ) hcl . for the enumeration of each stec 
 strain ( table 1 ) belonging to the different mixtures used for the inoculation 
 of the milk , viable cell densities were determined by spiral plating appropriate dilutions 
 in ts onto lb - a plates containing rifampicin ( 100 g ml ) , 
 spectinomycin ( 100 g ml ) , or nalidixic acid ( 40 g ml ) . lb - a plates were incubated at 37c for 24 hours and enumeration of the colonies was 
 performed with an automatic colony counter ec2 easy count 2 ( aes laboratory , 
 bruz , france ) . cheese 
 production was performed in a dairy laboratory in order to provide commercial 
 conditions during production . the chemical composition of the cheese samples 
 produced from the microfiltered milks is shown in table 3 . the fat and protein 
 content in the microfiltered milks were 37.00% and 32.58% w / w , respectively . it declined slightly to 6.03 ( sd : 0.02 ) during the first 4 hours ( draining of 
 curdled milk ) . then the ph decreased markedly from 6.03 to 4.65 ( sd : 0.05 ) at 
 the end of moulding step ( 24 hours ) . the ph remained almost stable until the 10th 
 day ( 4.64 to 4.75 ) ( sd : 0.02 ) and increased slightly over the last 10 days of 
 the ripening period to a ph of 5.11 ( sd : 0.03 ) ( table 4 ) . stec was never isolated from any of 
 the milk samples collected from the bulk storage tank nor from any of the 
 negative control cheeses . there were no differences between 
 the counts of nar and ar stec strains during the manufacture and ripening of 
 the microfiltered milk lactic cheeses . as an example , figure 2 shows the 
 survival of 4 stec mixtures : the ar2 ( 3 ar stec strains ) , as2 ( 3 nar stec 
 strains ) , arssb2 ( 2 nar and 1 ar stec strains ) , and asrra2 ( 2 ar and 1 nar stec 
 strains ) mixtures during cheese manufacture ( table 2 ) . in general , whatever the mixture 
 used , stec counts increased by a range of 1 to 2 2 log cfu g during the first steps of the cheese manufacturing and remained relatively 
 stable after salting until the drying stage of the cheeses . then , during 
 ripening ( 20 days ) , the counts of only one nar stec strain ( 346aspec ) 
 decreased to 10 cfu g. the other ar or nar stec strains were all 
 counted at levels ranging from 10 to 10 cfu g 
 ( figure 2 ) . at the end of ripening ( 20 days ) , 
 samples of inoculated cheeses were placed in simulated gastric fluid where the 
 numbers of surviving stec cells were assessed at 5 , 10 , 20 , 30 , 40 , 50 , 60 , 90 , 
 and 120 minutes . exposure to 
 sgf ( ph : 2.5 ) reduced the number of nar stec strains to undetectable levels within 
 40 , 50 , and 60 minutes for anr 42anal , anr 418arif-346aspec , and 360brif , respectively . in contrast to the nar stec 
 strains , all the ar stec strains survived an exposure of more than 120 minutes 
 in sgf at ph 2.5 ( table 5 ) . multiple applications of low levels 
 of acid stress during the life cycles of escherichia 
 coli o157:h7 and other pathogens might increase their likelihood for 
 survival in foods and may enhance the development and establishment of 
 stress - adapted strains with potentially increased virulence in food 
 environments [ 6163 ] . although acid tolerance in e. coli o157:h7 is normally transient , 
 being induced at low ph [ 14 , 30 , 64 , 65 ] , some outbreak strains ( e.g. , atcc 
 43895 ) have attained a permanently high , ph - independent acid resistance , probably because of an 
 evolutionary response to severe acid stress . the objective of the present study 
 was to investigate the growth and survival of ar and nar stec strains in 
 camembert - type cheeses . none of our findings have been subjected to a 
 statistical analysis since we have used mixtures of 3 stec strains for the 
 inoculation of the milk and , hence , some interaction between stec strains could 
 occur . the use of mixtures , instead of a single strain , is explained by the 
 limited number of batches ( 16 ) allowed by actilait and the decision to study 
 the kinetics of 8 different stec strains . moreover , it was not possible to 
 study pathogenic stec strains , such as e. 
 coli o157:h7 or e. coli o26 , due 
 to the lack of safety level / p3 laboratories . the number 
 of stec increased from 1 to 2 log10 at the beginning of the cheese 
 manufacture , whilst the ph decreased slightly from 6.45 ( milk ph ) to 6.03 
 ( first 4 hours ) . much of this initial increase could be attributed to the 
 entrapment of stec in the curd during coagulation followed by further 
 concentration during whey drainage . then we noted a plateau phase in the growth 
 of stec from the  cheese at the end of moulding  stage ( ph : 4.65 ) to the 
  cheese after drying  stage ( ph : 4.66 ) . from the middle of ripening ( 10 days , 
 ph : 4.75 ) to the end of ripening ( 20 days , ph 5.11 ) , the stec 
 population decreased markedly . in much the same way , after 24 hours of 
 manufacture and storage of camembert cheese inoculated at 10 cfu ml , 
 ramsaran et al .   observed an increase in e. coli o157:h7 counts of about 2 log10 . this increase was followed by a decrease in the counts of e. coli o157:h7 in all of the cheeses throughout ripening and 
 storage at 2c . .   showed that significant numbers of 
 viable e. coli o157:h7 could be 
 detected in raw goat milk lactic cheeses even 42 days after processing . in cheddar cheeses , aged for 60 and 120 days , and 
 stored at 7c , 
 the e. 
 coli o157:h7 population was reduced by less than 2 log . marek et al .   reported 
 that e. coli o157:h7 could persist in 
 unpasteurized cheddar cheese whey inoculated at 10 or 10 cfu ml for up to 2 - 3 weeks of storage at 4 , 10 , or 15c . the results of maher et al .   showed the presence of e. coli o157:h7 even after 90 days in 
 the rind and after 50 days in the core of smear ripened cheese produced from 
 raw milk . the acid adaptation response is a 
 phenomenon by which microorganisms show an increased resistance to 
 environmental stress after exposure to a moderate acid environment . in this 
 study , the acid adaptation of 4 nar stec strains was not induced by the slow 
 and mild acid conditions ( 4.654.75 ) found during the lactic cheese process 
 because these strains were rapidly destroyed during the strong acid exposure in 
 simulated gastric fluid ( ph 2.5 ) . hsin - yi and 
 chou   noted the same effects in fermented 
 milk but a completely opposite effect in acid fruit juice . the authors 
 explained that acid adaptation might lead to an increased susceptibility of the 
 test organism to antimicrobials , such as bacteriocins , hydrogen peroxide , 
 ethanol , and diacetyl , produced by lactic acid bacteria in milk products . the 
 effect of increasing susceptibility to these antimicrobials , due to acid 
 adaptation , may outweigh the effect of enhancing acid tolerance , reducing the 
 survival of acid - adapted e. coli o157:h7 in the milk products . observed that e. coli o157:h7 is able to induce an adaptive tolerance response ( atr ) 
 when exposed to mild acid conditions , thus conferring a higher resistance on 
 subsequent exposure to strong acid conditions . bergholz and whittam   studied the survival of enterohaemorrhagic escherichia coli of serotypes o157:h7 , 
 o26:h11 , and o111:h8 in a simulated gastric environment . their results 
 indicated that e. coli o157:h7 
 strains were better able to survive in a simulated gastric environment than the 
 stec strains belonging to the two other serogroups . the authors indicated that 
 this difference was reduced when cultures were held at stationary phase for 
 longer periods of time , suggesting that e. 
 coli o157:h7 cells rapidly achieve an enhanced state of ar in the early 
 stationary phase , an ability that may underlie the low infectious dose of this 
 pathogen . the present study indicates that ar 
 and nar stec strains , when initially present at 10 cfu ml in milk , would most likely survive artisanal camembert - type cheese manufacture 
 and ripening ( 20 days ) . the biggest decrease was observed for 
 an 
 nar stec strain ( 346aspec ) whose counts reached 10 cfu g at 20 days . even if we must keep in mind that the inoculation levels were 
 certainly higher than those observed in naturally contaminated milk , the low 
 infectious dose associated with pathogenic stec suggests that the 20 day 
 ripening period of these cheeses may not guarantee a safe product for consumers 
 if stec are present in the raw milk consequently , good milk hygiene is crucial 
 in order to reduce the risk of the presence of pathogens in the raw milk 
 cheeses . moreover , acid adaptation 
 of nar stec strains during the manufacture of cheeses prior to their exposure 
 to simulated gastric fluid did not increase the acid resistance of the bacteria . on the basis of these results , additional 
 investigations will be undertaken to evaluate the behavior of stec during the 
 manufacture of cheeses using a rapid curdling phase linked to a greater drop in 
 acidity than that employed in the present cheese technology .
growth and survival of acid - resistant ( ar ) and non - acid - resistant ( nar ) shiga - toxin - producing escherichia coli ( stec ) strains were investigated during the manufacture 
			 and ripening of microfiltered milk camembert cheeses . 
 the induction of acid resistance of the stec strains in cheeses 
			 was also studied . 
 six different mixtures of ar and/or nar stec strains were inoculated separately into microfiltered 
			 milk at a level of 103 cfu ml1 . the stec counts ( ar and nar ) initially increased by 1 to 2 log10 cfu g1 during cheese - making . 
 thereafter , the populations stabilized during salting / drying and then 
				 decreased during the early stages of ripening . exposing the stec strains in artificially inoculated cheeses 
				 to simulated gastric fluid ( sgf - ph : 2.0 ) reduced the number of nar strains to undetectable levels within 40 minutes , versus 120 minutes for the ar stec strains . 
 ar and nar stec were able to survive during the manufacture and ripening of camembert cheese prepared from microfiltered milk with no evidence of induced acid tolerance in nar stec strains .