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International Journal of Food Microbiology 45 (1998) 173–184

Influence of temperature and pH on survival of Escherichia coli

O157:H7 in dry foods and growth in reconstituted infant rice cereal

*Yun Deng, Jee-Hoon Ryu, Larry R. Beuchat

Center for Food Safety and Quality Enhancement , Department of Food Science and Technology, University of Georgia,

1109 Experiment Street , Griffin, GA 30223 -1797, USA

Received 9 June 1998; received in revised form 4 September 1998; accepted 21 September 1998

Abstract

Factors affecting the ability of Escherichia coli O157:H7 to survive in foods with a less than required for growth havew

not been fully defined. This study was undertaken to determine the ability of E . coli O157:H7 to survive in a commercial dry

infant rice cereal as affected by a (0.3560.04, 0.5260.03 and 0.7360.03), pH (4.0 and 6.8), and temperature (5, 25, 35w

and 458C), and in nine other reduced-a foods. Death of E . coli O157:H7 in cereal was enhanced with increased temperaturew

and decreased pH during a 16- to 24-week storage period. Survival was enhanced at pH 6.8 compared to pH 4.0 in cereal at

a 0.3460.04 during initial storage at 5 and 258C. The effect of temperature (8, 15, 21 and 308C) on survival and growth of w

acid-adapted cells of E . coli O157:H7 inoculated into cereal reconstituted with milk or apple juice at two inoculum levels(8.2–12.3 cfu/ml and 82–123 cfu/ml of slurry) was also studied. Growth occurred in cereal reconstituted with milk at all

test temperatures and in cereal reconstituted with apple juice at 15, 21 and 308C. Populations increased by . 1 log cfu/ml10

within 3–6 h at 21 and 308C. Acid-adapted and unadapted cells had similar growth patterns. The effects of temperature and

acid adaptation on survival of E . coli O157:H7 in nine commercial foods and food ingredients with pH 4.07–6.49 and aw

0.17–0.82 were determined. The pathogen survived in these foods for various lengths of time, depending the storage

temperature, with an order of survival of 58C. 218C. 378C. Survival appeared to be enhanced in foods with highest pH,

and acid-adapted cells retained higher viability than unadapted cells in only two of the nine test foods. Of particular

importance is the ability of E . coli O157:H7 to survive well in dry foods with a wide range in a and pH, particularly atw

refrigeration temperature. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Escherichia coli O157:H7; Dry foods; Water activity; Infant food

1. Introduction was first recognized as an important pathogen in the

early 1980s. The pathogen is known to cause

Escherichia coli O157:H7 has been associated hemolytic uremic syndrome, hemorrhagic colitis

with several outbreaks of foodborne disease since it cystitis, balanitis and thrombotic thrombocytopenic

purpura, especially in young children (Doyle et al.,* 1997). Although the major vehicle for E . coliCorresponding author. Tel.: 1 1-770-412-4740; Fax: 1 1-

770-229-3216; E-mail: [email protected] O157:H7 transmission is undercooked ground beef,

0168-1605/ 98/ $ – see front matter © 1998 Elsevier Science B.V. All rights reserved.

P I I : S 0 1 6 8 - 1 60 5 ( 9 8 ) 0 0 1 6 1 - 5



174 Y . Deng et al. / International Journal of Food Microbiology 45 (1998) 173 –184

other vehicles are chicken and lamb (Abdul-Raouf et ments by exposure to conditions similar to those in

al., 1996), raw milk (Ansay and Kaspar, 1997), and foods, then monitoring their survival and growth

pasteurized milk (Upton and Coia, 1994). Outbreaks characteristics, would provide valuable information

of infections have been associated with foods such as useful in predicting their behavior in food systems.

yogurt (Morgan et al., 1993), apple cider (Besser et Little is known about the survival of E . colial., 1993), deer jerky (Keene et al., 1997), and O157:H7 in dry foods or growth in reconstituted

salami (Centers of Disease Control and Prevention, infant foods. The study reported here was undertaken

1995). to determine the combined effects of a , pH andw

A study to determine carriage of E . coli O157:H7 temperature on the survival of E . coli O157:H7 in a

by persons living on dairy farms revealed elevated commercial dry infant rice cereal, and to determine

antibody titers against surface antigen of E . coli the effects of temperature and acid adaptation on

O157:H7 (Doyle et al., 1997). Outbreaks of E . coli survival and growth of the pathogen in cereal

O157:H7 infection have occurred in day-care centers reconstituted with milk or apple juice. In addition,

and nursing homes via person-to-person transmis- the effects of acid adaptation of E . coli O157:H7

sion, direct fecal–oral transmission, and cross-con- cells on their survival in commercial foods with and

tamination of foods, (Griffin and Tauxe, 1991). A food ingredients with reduced a

and pH werew

child infected with E . coli O157:H7 in a day care determined.

center-associated outbreak continued to excrete the

pathogen for 62 days (Doyle et al., 1997). The

pathogen has been detected in feces from a healthy2. Materials and methods

person (Rambach et al., 1997), which agrees with the

observation that infected animals may be asympto-2.1. Strains studied

matic (Cray and Moon, 1995). If these carriers work

in a day-care center, it is possible that cross-contami-Five strains of E . coli O157:H7 were used. Strain

nation of foods might occur, putting infants andE09 was isolated from an outbreak of infection

young children in danger.associated with ground beef, strains F500 and 932

Extensive studies have been done to determine

were isolated from human feces, strain 933 wasgrowth and survival characteristics of Salmonella isolated from beef, and strain 944 was isolated from(Foster and Hall, 1990) and enterohemorrhagic E .

salami.coli O157:H7 (Zhao et al., 1993; Leyer et al., 1995)

in acidic foods. Salmonella typhimurium has been

reported to be able to adapt to acid environments, 2.2. Preparation of media

thus greatly enhancing survival in low pH and other

harsh environments (Leyer and Johnson, 1992, 1997; Tryptic soy agar (TSA, pH 7.260.05, Difco,

Leyer et al., 1995). However, in these studies, acid Detroit, MI, USA), tryptic soy broth (TSB, pH

adaptation was achieved by adding HCl rather than 7.360.05, Difco), and sorbitol MacConkey agar

organic acids to cell suspensions to reduce the pH to (SMA, pH 7.0, Unipath-Oxoid, Hampshire, UK)

5.0–5.8. In natural food environments, bacteria are were prepared as specified by the manufacturers.

more often exposed to organic acids either produced Modified sorbitol MacConkey agar (MSMA, pH 7.0)by fermentation or added for the purpose of enhanc- was prepared by adding 4-methyl umbelliferyl-b-D-

ing sensory characteristics or preservation. Changes glucoronide (MUG, 0.2 g/ l) to SMA before heat

in pH are usually gradual. The remarkable ability of sterilization. Molten (47–508C) sterilized MSMA E . coli O157:H7 to tolerate acid environments was poured into petri dishes and held no more than

(Conner and Kotrola, 1995; Buchanan and Edelson, four days at 58C until used. Modified TSB (mTSB)

1996; Rowbury, 1997) has been demonstrated in a was prepared by combining (per liter of deionized

range of fermented and acidified foods (Hathcox et water) 30 g of TSB powder, 1.5 g of bile salts No. 3

al., 1995; Leyer et al., 1995; Miller and Kaspar, (Difco), 1.5 g of K HPO , 10 g of casamino acids2 4

1994; Zhao and Doyle, 1994; Zhao et al., 1993). (Difco), and 10 mg of acriflavin–HCl (Sigma, St.

Adapting E . coli O157:H7 cells to acidic environ- Louis, MO, USA). A stock solution (10 mg/ ml) of



Y . Deng et al. / International Journal of Food Microbiology 45 (1998) 173 –184 175

acriflavin was added (10 ml/ l) before sterilization at The pH of an aqueous slurry of the acidified cereal

1218C for 15 min. was 4.0, whereas the pH of the control cereal was

TSB supplemented with 1% glucose (TSBG) was 6.8. Dry cereal at each pH was deposited in

prepared by adding 10 g of glucose (Sigma) to 1 l of aluminum pans, placed over saturated solutions of

TSB. Both TSB and TSBG were dispensed into potassium acetate, magnesium nitrate, or potassiumeither 1503 16 mm screw-capped tubes (10 ml) or chloride, and held at 218C until desired a values

w

250-ml Erlenneyer flasks (100 ml) and autoclaved at were reached. The a of cereal was measured usingw

1218C for 15 min. Broths were stored at 58C for up an Aqualab CX2 instrument (Decagon Devices,

to four days until used. Pullman, WA, USA). Cereal (5 g) at each a / pHw

combination was placed in polypropylene vials and2.3. Preparation of inocula 50 ml of E . coli O157:H7 inoculum was added. An

additional 5 g of cereal was then added to each vial

Stock cultures of E . coli O157:H7 maintained on before caps were secured.

TSA (TSA containing 1.5% agar) slants at 58C were

transferred to 10 ml of TSB and incubated at 378C. 2.4.1. Enumeration of E . coli in dry cereal

Three consecutive 24-h loop transfers were made Populations of E . coli

O157:H7 in inoculatedbefore inoculating into 100 ml of TSB or TSBG. cereal were determined after incubation times rang-

Cultures incubated at 378C for 24 h were used to ing from 1 to 24 weeks. The content (10 g) of each

prepare inocula for use in the rice cereal study, vial was combined with 90 ml of sterile 0.1%

whereas 18-h cultures were used in studies involving peptone, pummeled in a stomacher for 15 s, and

survival and growth in infant rice cereal reconstituted surface plated in quadruplicate (0.25-ml samples) or

with milk or apple juice, and survival in low- and serially diluted in sterile 0.1% peptone and plated in

intermediate-a foods. duplicate (0.1-ml samples) on MSMA. Presumptivew

E . coli O157:H7 colonies were counted after in-2.3.1. Enumeration of E . coli O157: H 7 in inocula cubating plates at 378C for 48 h. Two or three

Cultures (24 h) of each strain of E . coli O157:H7 colonies from each sample were confirmed by ag-

were centrifuged for 15 min at 2100 g at 248C. The glutination using a latex agglutination test kit (Un-

supernatant was decanted and cell pellets were ipath-Oxoid) and by biochemical tests using the´resuspended in 25 ml of sterile 0.05 M potassium API-20E miniatured diagnostic kit (bio Merieux

phosphate buffer (pH 7.4). Populations of each strain Vitek, Hazelwood, MO, USA).

were determined by serially (1:10) diluting cell In cereal samples predicted to contain low popula-

suspensions in sterile 0.1% peptone water and sur- tions of E . coli O157:H7, the content of each vial

face plating (0.1 ml) duplicate samples on TSA and was combined with 90 ml of mTSB broth as well as

MSMA to confirm purity. Colonies were counted plated on MSMA. This mixture was incubated for

after incubation at 378C for 24 h. Cell suspensions 18–24 h at 378C, then streaked on MSMA, and again

were combined at ratios resulting in an inoculum incubated for 24 h at 378C. Plates were examined for

containing approximately equal populations of cells the presence of presumptive E . coli O157:H7

of each strain. colonies, which were subsequently subjected to latex

agglutination and biochemical tests for confirmation.2.4. Survival of E . coli O157: H 7 in dry rice

cereal 2.5. Growth and survival in reconstituted cereal

The combined effects of a , pH and temperature The ability of E . coli O157:H7 to grow in aw

on survival of E . coli O157:H7 in a commercial dry commercial infant rice food reconstituted with milk

infant rice cereal were determined. Dry cereal (a or apple juice as affected by temperature wasw

0.35) was placed in aluminum pans and further dried determined. Unadapted (control) and acid-adapted

in a forced-air oven at 508C for 18–20 h. Part of the cells were used as inocula.

cereal (3600 g) was combined with 75.6 g of Pasteurized milk (2% fat) purchased at a local

powdered citric acid, followed by thorough mixing. supermarket was adjusted to 218C within 1 h of




removal from refrigeration. Pasteurized apple juice mixture of either acid-adapted or control cell in-

was stored at 218C until used. Milk or apple juice oculum of E . coli O157:H7 was deposited on the

was added to dry rice cereal at a ratio of 6:1 (ml:g), surface of the sample. An additional 5 g of food was

based upon the cereal manufacturer’s recommenda- then added to each tube before caps were secured.

tion for reconstitution, and mixed with a sterile Sealed tubes were stored at 5, 21 and 378C.spatula. Cereal (200 g) reconstituted with 1200 ml of Populations of E . coli O157:H7 in the nine test

milk or apple juice at 218C was inoculated with 1 ml foods were monitored over a 12-week period. At

or 10 ml of a diluted mixed-strain suspension of E . each sampling time, the entire 10-g sample wascoli O157:H7 to give two population levels (0.91 placed into a stomacher bag with 90 ml of mTSB,

and 1.91 log cfu/ ml of slurry for acid-adapted pummeled for 1 min, and surface plated in quad-10

cells; 1.09 and 2.09 log cfu / ml for control cells) ruplicate (0.25 ml) or serially diluted in sterile 0.1%10

and stirred for 1 min. Samples (40 ml) of reconsti- peptone and plated in duplicate (0.1 ml) on MSMA.

tuted cereal were then deposited into sterile jars, Colonies were counted after incubating plates for 48

sealed, and immediately placed in incubators at 8, h at 378C. The remaining portion of the food slurry

15, 21 or 308C. was incubated at 378C for 18–24 h, then streaked

Reconstituted cereal was analyzed for populations into MSMA plates and incubated 24 h at 378C beforeof E . coli O157:H7 after 0, 3, 6, 9, 12, 24, 48 and 72 examining for the presence of E . coli O157:H7

h of storage. After shaking containers vigorously by colonies.

hand for 15 s, samples (1.0 ml) were surface plated Populations of aerobic mesophilic microorganisms

in quadruplicate (0.25 ml) or serially diluted in in nine test foods (Table 1) subsequently inoculated

sterile 0.1% peptone and surface plated in duplicate with E . coli O157:H7 were also determined. Samples

(0.1 ml) on MSMA. Colonies were counted after 48 (10 g) were combined with 90 ml of sterile 0.1%

h of incubation at 378C. The remaining portions of peptone water, pummeled for 1 min, and surface

samples were combined with 90 ml of sterile mTSB, plated (0.1 ml in duplicate) on plate count agar

incubated at 378C for 24 h, and streaked on mSMA (Difco). Plates were incubated at 308C f o r 4 8 h

to determine the presence of E . coli O157:H7. before colonies were counted.

2.6. Survival in foods with low and intermediate 2.7. Statistical analysisaw

Three replicates of each experiment were done.

Inocula were prepared as described for reconsti- All data were analyzed using the general linear

tuted cereal studies. Each test food (5 g) (Table 1) model of the Statistical Analysis System (SAS)

was placed in a sterile tube and 50 ml of a five-strain procedure. Duncan’s multiple range test was used to

Table 1

The pH, population of aerobic mesophilic microorganisms, and a of foods and food ingredients (listed in ascending order of initial a )w w

inoculated with E . coli O157:H7 and stored at 58C for up to 12 weeks

Aerobic

Food / mesophiles Storage time (weeks)

ingredient pH (cfu / 10 g) 0 1 2 3 5 8 12

Almond paste 5.93 , 1 0.82 0.82 0.81 0.82 0.82 0.81 0.83

Apple powder (unsulfured) 4.07 , 1 0.16 0.20 0.19 0.23 0.18 0.20 0.19

Apple powder (sulfured) 4.63 , 1 0.17 0.20 0.18 0.23 0.18 0.18 0.18

Buttermilk powder 6.49 22 0.21 0.26 0.27 0.30 0.35 0.37 0.38

Cheddar cheese seasoning 1 5.27 52 0.21 0.22 0.23 0.22 0.21 0.23 0.24

Cheddar cheese seasoning 2 5.62 44 0.32 0.31 0.32 0.36 0.31 0.34 0.33

Fig paste 4.25 370 0.70 0.68 0.69 0.69 0.70 0.70 0.70

Powdered chicken 6.43 , 1 0.37 0.35 0.35 0.36 0.34 0.39 0.38

Sour cream powder 5.26 10 0.29 0.31 0.31 0.31 0.30 0.31 0.31




determine if significant differences ( p# 0.05) ex- acidity on Salmonella newport and Staphylococcus

isted between populations detected in dry cereal at aureus in foods with reduced a was also noted byw

different a . The least significant difference test was Christian and Stewart (1973).w

used to determine if significant differences ( p# Death of E . coli O157:H7 was enhanced by an

0.05) existed between populations in dry cereal at increase in temperature, regardless of other testdifferent pH and between populations of acid- parameters. After one, five and 11 weeks of incuba-

adapted and non-adapted (control) cells detected in tion at 45, 35 and 258C, respectively, E . coli

dry foods. O157:H7 populations were below levels detectable

(, 10 cfu/g) by direct plating. The pathogen was

not detected in cereal stored at 458C for three weeks,

3. Results and discussion regardless of a or pH. Others have observed that E .w

coli O157:H7, at a given a , survives better inw

3.1. Survival in dry cereal salami (Clavero and Beuchat, 1996; Faith et al.,

1998a), pepperoni (Faith et al., 1998b), and bovine

The a of the infant rice cereal purchased at a feces (Wang et al., 1997) stored at refrigerationw

local supermarket ranged from 0.35 to 0.40. After temperatures compared to ambient temperatures.drying in a forced air oven, the cereal (a 0.18) was Calicioglu et al. (1997) observed that E . coliw

adjusted to desired a . The three a ranges O157:H7 inoculated into sausage incubated at 4 orw w

(0.3560.04, 0.5260.03 and 0.7360.03 ) of cereals 258C could be detected by enrichment after one

subsequently inoculated with E . coli O157:H7 were week. Some cells may have been injured by high

thus achieved via a water absorption process. E . coli temperature, low a and / or low pH, and entered aw

O157:H7 was not detected in uninoculated dry or viable but non-culturable state. These observations

equilibrated cereal. The addition of citric acid to underscore the importance of enrichment to detect

cereal (pH 6.7560.05) reduced the pH to 4.060.05. injured and low numbers of E . coli O157:H7 cells in

Results of studies to determine survival of E . coli foods that impose stress environments.

O157:H7 in cereal at six combinations of a and pHw

stored for up to 24 weeks at four storage tempera- 3.2. Growth and survival in reconstituted cereal

tures are shown in Table 2. The initial populations of E . coli O157:H7 were 6.31 and 6.10 log cfu / g in Populations of aerobic microorganisms in dry rice

10

inoculated cereal at pH 6.8 and 4.0, respectively. cereal, milk, and apple juice are listed in Table 3.

These populations were selected to simulate a situa- The population of aerobic mesophilic microorga-

tion where cross-contamination of cereal might occur nisms in pasteurized milk is similar to that reported

in the home or day-care center due to improper by Wang et al. (1997). E . coli O157:H7 was not

hygienic practices. The number of E . coli O157:H7 detected in dry cereal, pasteurized milk, or apple

in cereal subjected to all a , pH, and temperature juice.w

conditions decreased at least 1 log within one week Survival and growth characteristics of E . coli

of storage. At a given sampling time and storage O157:H7 in rice cereal reconstituted with milk or

temperature, survival of significantly higher popula- apple juice and held at 8, 15, 21 or 308C were

tions of E . coli O157:H7 was observed in cereal at determined. There were differences in pH values of higher a . This phenomenon was most evident the 18-h cultures of acid-adapted (TSBG, 4.860.2)

w

during the initial stage of storage at lower tempera- and unadapted (TSB, 6.260.1) cells used as inocula.

tures. This is in contrast to observations reported by Shown in Table 4 are populations of E . coli

Christian and Stewart (1973) and Juven et al. (1984) O157:H7 in reconstituted cereal inoculated with

that salmonellae survived better in dry foods as the acid-adapted (TSBG) [8.2 (0.91 log ) cfu/ ml] or10

a was decreased. control (TSB) [12.3 (1.09 log ) cfu /ml] cells. Tablew 10

Survival of E . coli O157:H7 was enhanced at pH 5 shows in reconstituted cereal inoculated with

6.8 compared to pH 4.0 in cereal at a 0.3560.04 higher populations of acid-adapted [82 (1.92 log )w 10

during storage for one week at 5 and 258C, and in cfu/ ml] and unadapted [123 (2.09 log ) cfu/ ml].10

cereal at all test a held at 358C. The lethal effect of Initial populations were determined by plating cellsw



Table 2

Population of E . coli O157:H7 in rice cereal as affected by a , pH, temperature and storage timew

aStorage Population (log cfu/g)10

temperature 1 week 2 weeks 3 weeks 4 weeks 5 weeks 7 weeks 8 weeks 9 weeks

b b(8C) a pH Log E Log E Log E Log E Log E Log E Log E Log Ew 10 10 10 10 10 10 10 10

5 0.356

0.04 6.8 b 3.93 a a 3.96 a b 3.55 c 2.88 a 4.0 c 3.46 b b 3.31 b b 3.40 c 3.27 c 3.06 a

0.5260.03 6.8 b 4.47 a a 4.55 b b 3.90 b 3.37 a

4.0 b 4.20 a a 4.81 a b 3.81 b 3.60 b 3.60 a

0.7360.03 6.8 a 5.07 a a 4.65 a a 4.70 a 4.52 a

4.0 a 4.77 a a 4.49 a a 4.39 a 4.68 a 4.41 a

25 0.3560.04 6.8 a 4.24 a b 3.11 a b 2.77 a b 2.44 a b 1.12 a

4.0 c 2.78 b b 2.20 b b 2.79 a b 2.03 a b 1.12 a

0.5260.03 6.8 b 3.30 a b 3.14 a b 2.78 a b 2.81 , 1.00 1

4.0 b 3.20 a b 2.16 b c 1.43 b , 1.00 b 1 , 1.00 1

0.7360.03 6.8 a 5.36 a a 5.04 a a 3.93 a a 3.80 a a 2.94

4.0 a 4.20 b a 3.77 b a 3.75 a a 3.28 b , 1.00 1

35 0.3560.04 6.8 b 3.12 a b 1.87 a , 1.00 1 1.52 1 , 1.00 1 , 1.00 1 , 1.00

4. 0 b 1 .70 b , 1.00 a 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00

0.5260.03 6.8 b 3.22 a , 1.00 1 , 1.00 1 , 1.12 1 , 1.00 1 , 1.00 2 , 1.00

4.0 b 2 .34 b , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 2 , 1.00

0.7360.03 6.8 a 4.58 a a 3.88 1.17 a , 1.00 1 , 1.00 1 , 1.00 2 , 1.00

4.0 a 3 .56 b , 1.00 1 1.67 a , 1.00 1 , 1.00 2 , 1.00 2 , 1.00

45 0.3560.04 6.8 , 1.00 , 1.00 2 , 1.00 2 , 1.00 2

4.0 , 1.00 1 , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2

0.5260.03 6.8 , 1.00 , 1.00 2 , 1.00 2 , 1.00 2

4.0 , 1.00 1 , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2

0.7360.03 6.8 , 1.00 , 1.00 2 , 1.00 2 , 1.00 2

4.0 , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

aLog cfu/ g dry cereal. Initial population (0 week) was 6.31 log cfu/ g of cereal at pH 6.8 and 6.10 log cfu/ g cereal at pH 4.0.Values

10 10 10

the same temperature, a and storage time) are significantly different ( p# 0.05). Values not preceded by the same letter, (within the saw

significantly different ( p# 0.05).b

Enriched in mTSB, 1 indicates E . coli O157:H7; 2 indicates E . coli O157:H7 was not detected.




Table 3 been associated with E . coli O157:H7 infectionThe pH and populations of aerobic mesophilic microorganisms in (Besser et al., 1993). The use of apple juice contami-dry cereal, milk and apple juice, and in cereal reconstituted with

nated with the pathogen to reconstitute infant ricemilk and apple juice

cereal may result in high populations within a shortItem pH Aerobic mesophiles

time.Rice cereal (dry) 6.8 , 10 cfu/g A previous study in our laboratory showed thatMilk 6.7 45 cfu / ml Bacillus cereus did not grow in infant rice cerealApple juice 3.8 , 1 cfu/ml

reconstituted with apple juice (Jaquette and Beuchat,Cereal reconstituted with milk 6.7 , 10 cfu/ml

1998). In the study reported here, E . coli O157:H7Cereal reconstituted with apple juice 4.6 , 10 cfu/ml

grew at 15, 21 and 308C, though at different rates. In

cereal reconstituted with apple juice and inoculated

on TSA and calculating log cfu / ml of reconstituted with 8.2–12.3 cfu of E . coli O157:H7 per ml (Table10

cereal. The number of viable E . coli O157:H7 cells 4), populations exceeded 1 log cfu / ml within 9, 2410

decreased within the first 3 h after inoculation, then and 72 h at 30, 21 and 158C, respectively.8

increased with time when cereal reconstituted with Populations of E . coli O157:H7 exceeding 10

milk was stored at 15, 21 or 308C, regardless of the cfu/ ml of cereal reconstituted with apple juiceinoculum population. caused no detectable change in color or aroma.

Acid-adapted and unadapted cells grew similarly However, this population in cereal reconstituted with

at the same temperature in cereal reconstituted with milk resulted in a sour aroma. Abdul-Raouf et al.

milk or apple juice. Unpasteurized apple juice has (1993) reported that the visual appearance of raw

Table 4

Populations of E . coli O157:H7 recovered from reconstituted rice cereal inoculated with acid-adapted (TSBG) cells [8.2 (0.91 log )10

cfu/ml] and unadapted (TSB) cells [12.3 (1.09 log ) cfu/ ml]10

aStorage Population (log cfu /ml)10

Reconstituted temperature Cell 3 h 6 h 9 h 12 h 24 h 48 h 72 h

bliquid (8C) type Log E Log E Log E Log E Log E Log E Log E 10 10 10 10 10 10 10

Milk 8 TSBG 0.93 a 1 0.54 1 , 0 1 , 0 1 0.30 a 1 , 0 1 , 0 1

TSB 0.40 b 1 , 0 1 , 0 1 , 0 1 0.65 a 1 0.54 1 0.18 1

15 TSBG 0.70 a 1 0.81 a 1 0.90 a 1 1.41 a 1 3.97 a 5.82 a 8.59 a

TSB 0.80 a 1 0.74 a 1 0.98 a 1 1.22 b 1 2.84 a 5.86 a 7.34 a

21 TSBG 0.30 a 1 1.41 a 1 2.05 a 1 3.18 a 6.48 a 8.44 a 8.67 a

TSB 0.88 a 1 1.31 a 1 1.67 a 1 2.51 a 5.76 a 8.73 a 8.08 a

30 TSBG 0.48 b 1 2.65 q 1 4.04 a 5.50 b 8.41 a 8.68 a 8.59 a

TSB 1.00 a 1 2.42 a 1 4.02 a 5.77 a 8.35 a 8.47 a 8.44 a

Apple juice 8 TSBG 0.65 1 0.65 a 1 0.18 a 1 , 0 1 0.70 a 1 , 0 1 , 0 1c

TSB , 0 1 1.02 a 1 0.48 a 1 0.18 1 0.18 a 1 , 0 1 0.18 1

15 TSBG , 0 1 0.30 a 1 0.40 a 1 0.30 a 1 , 0 1 0.93 a 1 1.41 a 1

TSB 0.48 1 0.81 a 1 0.40 a 1 0.00 a 1 , 0 1 1.00 a 1 1.65 a 1

21 TSBG 0.81 a 1 , 0 1 , 0 1 0.88 a 1 1.82 a 1 3.80 a 1 5.30 a

TSB 0.40 a 1 0.18 1 , 0 1 0.65 a 1 1.76 a 1 3.96 a 1 4.76 a

30 TSBG , 0 1 0.30 a 1 1.11 a 1 2.17 a 1 4.41 a 1 6.77 b 8.25 a

TSB , 0 1 0.48 a 1 1.40 a 1 2.20 a 1 4.78 a 1 7.13 a 8.22 a

aValues not followed by the same letter (within the same reconstitution fluid, temperature and storage time) are significantly different

( p# 0.05).b

Indicates the presence of E . coli O157:H7 detected by enrichment in mTSB.c, 1 cfu/ml.




Table 5

Populations of E . coli O157:H7 recovered from reconstituted rice cereal inoculated with acid-adapted (TSBG) cells [82 (1.91 log ) cfu / ml]10

and unadapted (TSB) cells [123 (2.09 log ) cfu/ ml]10

aStorage Population ( log cfu / ml)

10

Reconstituted temp. Cell 3 h 6 h 9 h 12 h 24 h 48 h 72 hb

liquid (8C) type Log E Log E Log E Log E Log E Log E Log E 10 10 10 10 10 10 10

Milk 8 TSBG 1.26 a 1 1.41 a 1 1.10 b 1 1.24 b 1 0.98 b 1 0.90 a 1 0.78 a 1

TSB 1.44 a 1 1.35 a 1 1.45 a 1 1.41 a 1 1.32 a 1 1.19 a 1 0.88 a 1

15 TSBG 1.41 a 1 1.26 b 1 1.57 b 1 2.01 a 1 3.87 a 6.61 a 7.90 a

TSB 1.27 a 1 1.62 a 1 1.78 a 1 2.20 a 1 3.74 a 7.10 a 8.31 a

21 TSBG 1.35 a 1 1.89 b 1 2.44 a 3.41 a 5.63 a 8.81 a 8.90 a

TSB 1.57 a 1 2.11 a 1 2.69 a 3.62 a 6.70 a 8.69 a 8.96 a

30 TSBG 1.60 a 1 2.03 b 4.67 a 5.44 a 8.62 a 8.89 a 8.78 a

TSB 1.59 a 1 3.32 a 4.95 a 6.56 a 8.50 a 8.69 a 8.49 a

Apple juice 8 TSBG 1.36 a 1 1.24 a 1 1.51 a 1 1.31 a 1 1.16 a 1 1.15 a 1 0.81 a 1TSB 1.23 a 1 1.54 a 1 1.45 a 1 1.24 a 1 0.90 a 1 1.00 a 1 0.54 a 1

15 TSBG 1.41 a 1 1.13 a 1 1.37 a 1 1.34 a 1 1.08 a 1 1.45 b 1 2.06 a 1

TSB 1.13 a 1 1.29 a 1 1.45 a 1 1.37 a 1 1.04 a 1 1.98 a 1 2.84 a 1

21 TSBG 1.18 a 1 1.26 a 1 1.20 a 1 1.45 b 1 2.58 a 1 4.43 a 1 5.81 a

TSB 1.18 a 1 1.27 a 1 1.27 a 1 1.73 a 1 2.90 a 1 5.02 a 1 6.22 a

30 TSBG 1.52 a 1 1.76 a 1 1.88 b 1 2.81 b 1 5.08 a 1 6.62 a 8.18 a

TSB 1.36 b 1 1.85 a 1 2.24 a 1 3.25 a 1 6.10 a 1 8.13 a 8.22 a

aValues not followed by the same letter (within the same reconstitution fluid, temperature and storage time) are significantly different

( p# 0.05).b

Indicates the presence of E . coli O157:H7 detected by enrichment in mTSB.

vegetables was not influenced substantially by pathogen grew at 15, 21 and 308C, and survived for

growth of E . coli O157:H7. 72 h at 88C in cereal reconstituted with apple juice.

Populations of E . coli O157:H7 in reconstituted This further confirms observations that E . coli

cereal initially inoculated with 1.91 or 2.09 log O157:H7 has a high tolerance to the acidic (pH 4.6)10

cfu/ ml of acid-adapted or unadapted cells, respec- conditions.

tively, are shown in Table 5. In cereal reconstituted

with milk, the population of E . coli O157:H7 3.3. Survival of E . coli O157: H 7 in low- and

increased at all incubation temperatures except 88C intermediate-a foodsw

as occurred with cereal inoculated with the lower

inoculum (Table 4). At 88C, the population slowly Survival of acid-adapted and unadapted E . coli

declined during 72 h of incubation. As with cereal O157:H7 cells inoculated into nine low- andinoculated lower numbers of E . coli O157:H7, intermediate-a foods and stored at 5, 21 and 378C

w

growth of the higher inoculum occurred at 15, 21 for up to 12 weeks was determined. Populations of

and 308C. The lag phase of growth in cereal with the aerobic mesophilic microorganisms, pH and a of w

higher inoculum was shorter than the lag phase in foods and food ingredients are shown in Table 1.

cereal containing the lower inoculum. Cereal recon- The pH of test foods ranged from 4.07 to 6.49 and

stituted with apple juice did not change in color and the a ranged from 0.16 to 0.82.w

aroma. At a given storage time, increased storage Shown in Table 6 are the populations of acid-

temperature enhanced the growth of E . coli adapted and unadapted E . coli O157:H7 cells in test

O157:H7, with no marked differences in growth foods as affected by storage temperature. Initial (0

rates of acid-adapted and unadapted cells. The week) populations were determined by plating the




Table 6

Populations of E . coli O157:H7 (log cfu/ ml) in foods and ingredients as affected by cell type (acid-adapted, grown in TSBG; unadapted,10

grown in TSB), temperature and time

aStorage Population (log cfu /g)10

Food/ temperature Cell 1 week 2 weeks 3 weeks 5 weeks 8 weeks 12 weeks 19 weeks

bingredient (8C) type Log E Log E Log E Log E Log E Log E Log E 10 10 10 10 10 10 10

Almond paste 5 TSBG 4.15 b 3.99 b 4.26 b 4.32 a 4.00 a 3.83 a

TSB 4.34 a 4.20 a 4.40 a 4.42 a 3.98 a 3.76 a

21 TSBG 3.20 b 2.16 a 1 2.18 a 1 , 1.00 1 , 1.00 1 , 1.00 2

TSB 3.58 a 2.33 a 1 2.15 a 1 , 1.00 1 , 1.00 1 , 1.00 2

37 TSBG , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2

Apple powder 5 TSBG 3.89 a 3.83 a 3.90 a 3.71 b 3.25 a 2.51 a

(unsulfured) TSB 3.48 b 3.40 a 3.85 a 4.03 a 3.18 a 2.39 b

21 TSBG 2.88 a 1.78 a 1 2.22 a 1 1.30 a 1 , 1.00 1 , 1.00 1TSB 2.59 b 1.85 a 1 2.32 a 1 1.18 a 1 , 1.00 1 , 1.00 1

37 TSBG , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

Apple powder 5 TSBG 3.34 a 3.37 a 3.92 a 3.86 b 3.42 b 2.90 b

(sulfured) TSB 3.65 b 3.65 a 3.86 a 4.41 a 4.08 a 3.68 a

21 TSBG 3.35 a 2.15 a 2.81 a 1 1.65 a 1 1.60 a 1 , 1.00 1

TSB 3.12 b 2.49 a 3.18 a 1 1.98 a 1 2.52 a 1 1.30

37 TSBG , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

Buttermilk powder 5 TSBG 3.83 a 4.33 a 4.13 a 4.48 a 4.00 a 3.58 aTSB 4.20 a 3.81 b 4.50 a 3.99 b 3.54 b 4.00 a

21 TSBG 3.88 a 2.77 a 1 2.37 a 1 2.57 a 1 1.88 a 1 , 1.00 1

TSB 3.60 a 2.53 a 1 2.33 a 2.54 a 1 1.54 a 1 , 1.00 1

37 TSBG 1.60 a 1 , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB 1.48 a 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 2

Cheddar cheese 5 TSBG 4.27 a 4.11 a 4.18 a 3.88 a 4.25 a 3.13 a

seasoning 1 TSB 3.80 b 3.34 b 3.50 b 3.55 b 3.09 b 2.46 a

21 TSBG 3.16 a 2.52 a 1 2.37 a 1 1.88 1 , 1.00 1 , 1.00 1

TSB 2.62 b 1.60 b 1 1.95 b 1 , 1.00 1 , 1.00 1 , 1.00 1

37 TSBG , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

Cheddar cheese 5 TSBG 4.51 a 4.00 a 3.69 a 3.49 a 3.05 a 2.18 a

seasoning 2 TSB 3.45 b 3.11 b 3.33 b 2.90 b 2.18 b 1.15 b

21 TSBG 2.41 a 1.48 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1

TSB 1.48 b , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1

37 TSBG , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2




Table 6. Continued

aStorage Population (log cfu /g)10

Food/ temperature Cell 1 week 2 weeks 3 weeks 5 weeks 8 weeks 12 weeks 19 weeks

bingredient (8C) type Log E Log E Log E Log E Log E Log E Log E 10 10 10 10 10 10 10

Fig paste 5 TSBG 4.11 a 3.45 b 2.82 a , 1.00 2 , 1.00 2 , 1.00 2

TSB 4.23 a 3.67 a 3.01 a , 1.00 2 , 1.00 2 , 1.00 2

21 TSBG , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

37 TSBG , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

Powdered chicken 5 TSBG 4.21 a 4.24 a 4.37 a 4.29 a 3.97 a 4.10 a

TSB 4.33 a 4.20 a 4.19 a 4.25 a 4.16 a 3.55 a

21 TSBG 3.46 a 3.00 a 1 2.99 a 1 2.98 a 1 1.78 b 1 , 1.00 1

TSB 3.57 a 3.02 a 1 3.01 a 1 2.66 a 1 2.51 a 1 , 1.00 1

37 TSBG , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 2 , 1.00 2

TSB 1.48 1 , 1.00 1 , 1.00 1 , 1.00 1 , 1.00 2 , 1.00 2

Sour cream 5 TSBG 4.20 a 3.99 a 3.69 b 3.48 b 2.16 b 2.26 b

powder TSB 4.45 a 3.80 b 4.43 a 4.18 a 3.66 a 2.94 a

21 TSBG 2.34 b 2.65 a 1 , 1.00 a 1 , 1.00 1 , 1.00 1 , 1.00 2

TSB 3.26 a 2.32 b 1 2.29 a 1 1.18 1 , 1.00 1 , 1.00 2

37 TSBG , 1.00 2 , 1.00 2 , 1.00 2 , 1.00 2

TSB , 1.00 1 , 1.00 2 , 1.00 2 , 1.00 2

aValues not followed by the letter (within the same food and storage temperature) are significantly different ( p# 0.05). Initial populations of

acid-adapted and unadapted cells were 5.48 and 5.71 log cfu/ g, respectively, in almond paste, Cheddar cheese seasonings and apple10

powder, and 5.60 and 5.83 log cfu/ g, respectively, in fig paste, chicken powder, sour cream powder and buttermilk powder.10

b Enrichment in mTSB: 1 indicates presence of E . coli O157:H7; 2 indicates that E . coli O157:H7 was not detected.

inoculum on TSA and then calculating cfu/ g of exception of fig paste, in which E . coli O157:H7

food. The initial populations of acid-adapted and survived three weeks but not eight weeks, the

unadapted cells were 5.48 and 5.71 log cfu/ g, pathogen maintained high viability for 19 weeks.10

respectively, in almond paste, Cheddar cheese Death in fig paste is attributed to stress conditions

seasonings, and apple powders, and 5.60 and 5.83 imposed by low pH and a relatively high a at whichw

log cfu / g, respectively, in fig paste, chicken pow- metabolic activity of cells would not be minimized.10

der, sour cream powder, and buttermilk powder. Numbers of E . coli O157:H7 recovered from sul-

With the exceptions of buttermilk powder and pow- fured and unsulfured apple powder did not differ

dered chicken, E . coli O157:H7 was not detected in significantly.

foods stored at 378C for two weeks. The pathogen Various types and amounts of solute in test foodssurvived in the latter foods for at least eight and five may influence survival of E . coli O157:H7. The type

weeks, respectively. Of the nine products tested, of solute is known to influence minimum a atw

buttermilk powder and chicken powder had the which E . coli O157:H7 will grow (Buchanan and

highest pH values (Table 1), suggesting that acid Bagi, 1997), and may also influence the rate of

environments adversely affected viability. The same inactivation at a values less than required forw

trend was noted for rice cereal (Table 2). At 218C, E . growth. Our observation that E . coli O157:H7 sur-

coli O157:H7 survived for less than two weeks in fig vives better in reduced-a foods at refrigerationw

paste, eight weeks but not 12 weeks in almond paste temperature agrees with observations on its behavior

and sour cream powder, and at least 12 weeks in all in dry rice cereal and reports by Clavero and Beuchat

other foods. When storage was at 58C, with the (1996) indicating that survival was better in salami




as a simple means of evaluating the acid tolerance of at 58C than at 208C or 308C and Zhao and Doylestationary-phase cells. Appl. Environ. Microbiol. 62, 4009–(1994) that E . coli O157:H7 survived in mayonnaise4013.

for 34 to 55 days at 58C and for 8 to 21 days at 208C.Buchanan, R.L., Bagi, L.K., 1997. Effect of water activity and

Acid-adapted cells consistently retained higher humectant identity on the growth kinetics of Escherichia coli

viability than control cells in Cheddar cheese season- O157:H7. Food Microbiol. 14, 413–423.Calicioglu, M., Faith, N.G., Buege, D.R., Luchansky, J.B., 1997.ings stored at 5 and 218C. This suggests that acid

Viability of Escherichia coli O157:H7 in fermented semidryadaptation, at least adaptation to lactic acid, resultedlow-temperature-cooked beef summer sausage. J. Food Prot.

from growth in TSBG, rendering cells more tolerant60, 1158–1162.

to lactic acid presumed to be present in these dairy Clavero, M.R.S., Beuchat, L.R., 1996. Survival of Escherichia

products. In a previous study (Deng et al., 1998) we coli O157:H7 in broth and processed salami as influenced by

pH, water activity, and temperature and suitability of mediaobserved that enhanced acid tolerance of acid-for its recovery. Appl. Environ. Microbiol. 62, 2735–2740.adapted E . coli O157:H7 cells was influenced by

Centers of Disease Control and Prevention 1995. Escherichia colistrain, type of acidulant used to induce acid adapta-

O157:H7 outbreak linked to commercially distributed dry-tion, and the nature of secondary stresses. Studies by cured salami-Washington and California. Morbid. Mortal.

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Christian, J.H.B., Stewart, B.J., 1973. Survival of Staphylococcus

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