# Growth and Survival of Acid-Resistant and Non-Acid-Resistant Shiga-Toxin-ProducingEscherichia coli Strains during the Manufacture and Ripening of Camembert Cheese

Authors: M. P. Montet; E. Jamet; S. Ganet; M. Dizin; S. Miszczycha; L. Dunière; D. Thevenot; C. Vernozy-Rozand
Journal: International Journal of Microbiology
 (2009)

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## Abstract

Growth and survival of acid-resistant (AR) and non-acid-resistant (NAR) Shiga-toxin-producingEscherichia 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 mL−1. The STEC counts (AR and NAR) initially increased by 1 to 2 log10 CFU g−1 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.


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## Body

## 1. Introduction

Shiga-toxin- (Stx-) producingEscherichia 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 [3–6] or the conditions in acidic foods [7–9].
It is noteworthy that acid tolerance plays a vital role in the survival and
virulence of diarrheagenic E. coli strains [10–12]. The ability of E. coli O157:H7 strains to survive in
acidic conditions has been studied extensively [13–16], 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 [19–22]. Cattle
are considered to be a major reservoir of E.
coli O157:H7 for human infection [23]. The pathogen has been isolated
from raw milk [24], 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 [27], but total inactivation was
obtained during heat treatment (57°C 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 1CFUmL−1.  E. coli O157:H7 also survived the manufacture and storage of Camembert and Feta cheeses
at 2±1°C for 65 and 75 days, respectively [28].Moreover, this pathogen was able to
survive all stages of smear-ripened cheese production for up to 70 days
postmanufacture [29].E. coli O157:H7 was not eliminated from goat's milk lactic cheese, made with raw milk,
because this organism tolerates the low pH [30] and low temperatures [31, 32] which characterize the
manufacturing and ripening process.Surprisingly, studies on contamination
of milk, or its products, with non-O157E. 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 [35–37] and, very recently, a study
described the prevalence of STEC in Swiss raw milk cheeses [38].  Moreover, there are no published
experiments evaluating the growth or survival of non-O157 STEC in cheeses.However, non-O157E. 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 [39]. For example, in Italy [40], Denmark [41], and Germany [42], 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 [43]. Other human infections with
non-O157 STEC from dairy products are
well documented [44–47]. 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 [48]. A similar phenomenon, termed the
“acid tolerance response,” has been demonstrated in S. typhimurium  [49, 50].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 ofE. 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 [51].The main objectives of the present
study were firstly to evaluate the growth and survival of AR and NAR Shiga-toxin-producingE. coli strains and, secondly, to
check whether microfiltered milk Camembert cheeses could induce acid tolerance
in inoculated NAR STEC strains.


## 2. Materials and Methods

## 2.1. Bacterial Strains and Culture Conditions

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, 52–54].With the aim of evaluating the
ability of these bacteria to survive exposure to acid, we used the protocol
described by Castanie-Cornet et al. [10], in which three AR mechanisms were
tested. These mechanisms are as follows:(i)
AR1: oxidative
system (glucose–repressed system),(ii)
AR2: system
depending on the presence of glutamate,(iii)
AR3: system
depending on the presence of arginine. A fourth mechanism is a fermentative
acid resistance system. It was tested according to the method previously
described by Large et al.  [55].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 Table1. 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 μg/mL) in increasing concentrations, using the protocol
described by Truong et al. [56]. Three rifampicin and three
spectinomycin mutants were selected using the same protocol. 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) were isolated. We carefully checked that the chosen resistant
derivatives were neither affected in their growth rate nor in their
acid-resistance properties (data not shown).Table 1
Virulence factors and acid
resistance of the STEC strains used for the artificial contamination of milk.
STEC strainsVirulence factorsAcid-resistanceReferenceStrainsSerotypesaOriginseaegenecstx1  genecstx2  genecShiga-toxin producingbAR1AR2AR3AR4rate of survival (%)rate of survival
(%)rate of survival
(%)rate
of survival
(%)AR
STEC strainsANRO6:H10Raw milkNPPP960,500Vernozy-Rozand
et al. [34]415ASpeccheeseANROntH8Raw milkNPPP607600Vernozy-Rozand et al. [34]245A1RifcheeseANRO166:H28EnvironmentNNPP80100Vernozy-Rozand et al. [54]V1SpecANRO11:H43EnvironmentNPNP67000Vernozy-Rozand et al. [54]V10NalNAR STEC strainsANRO6:H1Raw milkNPPP0000Vernozy-Rozand et al. [34]360BRifcheeseANRO6:H1Raw milkNPPP0,7000,2Vernozy-Rozand et al. [34]42ANalcheeseANRO6:H10Raw milkNPNP0000Vernozy-Rozand et al. [34]418ARifcheeseANRO174:H8Raw milkPPPP0000Vernozy-Rozand et al. [34]346ASpeccheeseaOnt: O type not corresponding to any serogroup
between O1 and O 174.
bP production.
cP,
positive.
N, negative.
AR1: oxidative system
(glucose-repressed
system), AR2: system depending on the presence of glutamate, AR3: system
depending on the presence of arginine, AR4: system depending on the presence of
lysine.
Rif:
Rifampicin resistance, Spec: Spectinomicin resistance, Nal: Nalidixic acid resistance.


## 2.2. Microfiltered Cow Milk

Raw cow milk samples were collected
aseptically from the bulk storage tank after it had been cooled to <5°C
and maintained refrigerated at 3°C for transportation to Actilait. Cheeses were made with raw cow
milk standardized [57] by microfiltration. Raw milk was
microfiltered at  40°C through a 1.4μm 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. [58].


## 2.3. Inoculation Procedure and Preparation of the STEC Mixtures

Cultures of
antibiotic-resistant STEC strains were maintained at−80°C 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 37°C. Strains were grown
individually at 37°C for 24 hours in LB broth, with or without antibiotics, to reaffirm that the
strains retained resistance. All overnight cultures were centrifuged at 8000×g for 10 minutes. Cells were washed twice in 0.1% peptone water and suspended
in 0.1% peptone water to achieve the required inoculation (106CFUmL−1) level by standardization of the absorbance at A600nm using a spectrophotometer (BioPhotometer, Eppendorf, Le Pecq, France). Mixtures
were prepared by combining the 3 adequate STEC strains in equal amounts. 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.Table 2
Composition
of the STEC strains mixtures used for the inoculation of milk.
Cocktail
denominationCocktail compositionSTEC strainsAR1-AR23 AR STEC strainsANR
V10Nal-ANR 245A1Rif-ANR 415ASpecAS1-AS23 NAR STEC strainsANR 42ANal-ANR 360BRif-ANR 346ASpecARSSa1-ARSSa21 AR STEC strainsANR V10Nal2 NAR STEC strainsANR 360BRif-ANR 346ASpecARSSb1-ARSSb21 AR STEC strainsANR V1Spec2 NAR STEC strainsANR 42ANal-ANR 360BRifASRRa1-ASRRa22 AR STEC strainsANR
245A1Rif-ANR 415ASpec1 NAR STEC strainsANR
42ANalASRRb1-ASRRb22 AR STEC strainsANR V10Nal-ANR 415ASpec1 NAR STEC strainsANR 418ARifThe STEC population in each mixture
was confirmed by plating100μL 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 103CFUmL−1 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).


## 2.4. Cheese Manufacture

Artisanal microfiltered cow's milk
lactic cheeses were prepared following the industrial specifications of the
French Institute of cheese (Actilait).The milk
was inoculated (2×1011CFU per 100 kg) with mesophilic
starters MM100 (Lactococcus lactis subsp. lactis,  cremoris, and lactis biovar diacetylactis), (Rhodia,
Dangé-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 32°C and renneting was
carried out using 30 mL of rennet per 100 L of
milk (at 530mgL−1  of
chymosin, Berthelot, ABIA S.A. Meursault,
France). 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 77×110mm cylindrical plastic moulds. Cheeses drained for 20–22
hours at 20°C were turned twice during this time. Cheeses weighing approximately 250 g were removed from the
mould, and the temperature of the cheese-making chamber was reduced to 20–22°C. After 20 hours, the
cheeses were plunged into a saturated brine solution at 10°C for 25 minutes. Cheeses
were dried at 13°C for 5 hours and matured at 11°C and 95% relative humidity for 20 days, then finally packaged and stored at 4°C.The manufacturing protocol for lactic cheeses
made with microfiltered is outlined in Figure1.Figure 1
Flowchart for the manufacture of lactic cheeses
made with microfiltered milk.


## 2.5. Sampling Steps

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).


## 2.6. Microbiological Analysis

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, bioMérieux, 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(100μgmL−1), spectinomycin (100μgmL−1), or nalidixic acid (40μgmL−1), when
appropriate, and incubated for 24 hours at 37°C. Enumeration of the
colonies was performed with an automatic colony counter EC2 easy count 2 (AES
Laboratory, Bruz, France).


## 2.7. Physical-Chemical Measurements

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
(Précisa XM60), according to AFNOR (NF V 04-282), and the cheese fat content
was measured by the Heiss butyrometric method
[59]. 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).


## 2.8. Exposure of STEC, Present in Artificially Inoculated Cheeses, to Simulated Gastric Fluid (SGF)

Simulated gastric fluid was prepared accorded to the
protocol used by Yuk and Marshall
[60]. More precisely, simulated gastric fluid
consisted of 8.3gL−1 of proteose-peptone (Fluka-Biochemika, Switzerland), 3.5gL−1 of
D-glucose (Fluka-Biochemika, Switzerland), 2.05gL−1 of
NaCl (Sigma-Aldrich, Lyon, France), 0.6gL−1 of KH2PO4 (Merck Sharp
& Dohme, Paris, France), 0.11gL−1 of CaCl2 (Sigma-Aldrich, Lyon, France),
0.37gL−1 of KCl (Sigma-Aldrich, Lyon, France), 0.05gL−1 of ox
bile (Sigma-Aldrich, Lyon, France), 0.1gL−1 of lysozyme
(Sigma-Aldrich, Lyon, France), and 13.3mgL−1 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), at37°C, 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 (Table1) belonging to the different mixtures used for the inoculation
of the milk, homogenate cheese samples were taken at regular time intervals.
Viable cell densities were determined by spiral plating appropriate dilutions
in TS onto LB-A plates containing rifampicin  (100μgmL−1),
spectinomycin (100μgmL−1), or nalidixic acid (40μgmL−1).
LB-A plates were incubated at  37°C for 24 hours and enumeration of the colonies was
performed  with an automatic colony counter EC2 easy count 2 (AES Laboratory,
Bruz, France).


## 3. Results

## 3.1. PH and Physicochemical Properties during Manufacture and Ripening of The Cheeses

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 Table3. The fat and protein
content in the microfiltered milks were 37.00% and 32.58% w/w, respectively.Table 3
Physicochemical
properties of Camembert cheeses.
ComponentCheese at the end
of moulding (day 1)Cheese at the end of ripening (day 20)Dry matter (% w/w)38.37±0.4942.02±0.54Fat content (% w/w)19.44±0.3121.25±0.29Fat on dry matter (% w/w)50.65±0.5550.58±0.40Moisture content (% w/w)76.53±0.4373.65±0.50Salt content (% w/w)2.58±0.13pH4.66±0.055.11±0.03Average values in 4 uninoculated cheeses as negative
controls.
Mean± standard deviation.The pH of the milk was6.45±0.02.
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).Table 4
Changes in pH values during
cheese processing.
Stages of samplingMilk
before maturationCurd
during drainingCheese at
the end of mouldingCheese
after saltingCheese after dryingCheese at
the middle of ripening (D+10)Cheese at
the end of ripening (D+20)Inoculated cheeses6.45±0.026.03±0.024.65±0.054.64±0.044.66±0.054.75±0.025.11±0.03Uninoculated cheeses6.44±0.036.02±0.044.64±0.064.63±0.064.65±0.054.73±0.045.10±0.02pH values for
each cheese, inoculated or not, and at each time of sampling (i.e., 10 pH
measures per time of sampling).
Mean ± standard deviation.


## 3.2. Survival of STEC during Lactic Cheese Manufacture

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, Figure2 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).Counts of
STEC strains during the cheese manufacture. AR2: 3 AR STEC strains, AS2: 3 NAR
STEC strains, ARSSb2: 1 AR and 2 NAR STEC strains, ASRRa2: 2 AR and 1 NAR STEC
strains. AR strains: black lines, symbols:▵, ◯, ×. NAR strains: grey lines, symbols:  ▿,  ⊠,  ⋄. Step 1: milk prior to maturation,
Step 3: cheese at moulding stage, Step 5: cheese after drying, Step 7: cheese
at the end of ripening (20 days).
(a)(b)(c)(d)In general, whatever the mixture
used, STEC counts increased by a range of 1 to 22logCFUg−1 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 10CFUg−1. The other AR or NAR STEC strains were all
counted at levels ranging from 102 to 104CFUg−1
(Figure 2).


## 3.3. Survival of STEC in Simulated Gastric Fluid, PH 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 ANR42ANal, 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).Table 5
Survival
rates of STEC strains in simulated gastric fluid at pH: 2.
Acid-resistanceStrainSurvival rate (%)aTime (min)T(5)T(10)T(20)T(30)T(40)T(50)T(60)T(90)T(120)ARANR 415ASpec95957160503224104ANR 245A1Rif867565634643361310ANR V1Spec978013011511010810510034ANR V10Nal81747166564929188NARANR 360BRif3513250.370.180.03≤0.01≤0.01≤0.01ANR 42ANal55366.670.31≤0.01≤0.01≤0.01≤0.01≤0.01ANR 418ARif66417.180.400.14≤0.01≤0.01≤0.01≤0.01ANR 346ASpec26275940.59≤0.01≤0.01≤0.01≤0.01aPercentage of survival calculated as   100× the
number of CFU per gram remaining
after the acid treatment divided by the initial CFU per gram at time zero.


## 4. Discussion

Multiple applications of low levels
of acid stress during the life cycles ofEscherichia
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 [61–63]. 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 [17], 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 asE.
coli O157:H7 or E. coli O26, due
to the lack of safety level/P3 laboratories.The number
of STEC increased from 1 to2log10 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 104CFUmL−1,
Ramsaran et al. [28] observed an increase in E. coli O157:H7 counts of about 2log10.
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 2°C.Vernozy-Rozand et al. [51] 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 7°C,
the E.
coli O157:H7 population was reduced by less than 2 log [66]. Marek et al. [67] reported
that E. coli O157:H7 could persist in
unpasteurized Cheddar cheese whey inoculated at 105 or 102CFUmL−1 for up to 2-3 weeks of storage at 4, 10, or 15°C. The results of Maher et al. [29] 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 (4.65–4.75) 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 [68] 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. Jordan et
al.  [30] 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 [69] 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 at103CFUmL−1 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 10CFUg−1 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.



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*Source: 653481-2009-02-11.xml*