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The Effect of NaCl on Survival of Shigella

The Effect of NaCl on Survival of Shigella
The Effect of NaCl on Survival of Shigella

774

Journal of Food Protection,Vol.65,No.5,2002,Pages774–779

The Effect of NaCl on Survival of Shigella exneri in Broth as Affected by Temperature and pH??

LAURA L.ZAIKA*

Microbial Food Safety Research Unit,U.S.Department of Agriculture,Agricultural Research Service,Eastern Regional Research Center,

600East Mermaid Lane,Wyndmoor,Pennsylvania19038,USA

MS01-367:Received26September2001/Accepted5January2002

ABSTRACT

Shigella,a major foodborne pathogen,survives well in salt-containing environments.However,systematic data are scarce.

We studied the behavior of Shigella exneri5348in brain heart infusion broth(pH4to6)containing0.5to8%NaCl.

Stationary-phase cells were inoculated into sterile media at initial concentrations of6to7log10CFU/ml and incubated at12 to378C.Bacterial population sizes were determined periodically by plate counts.Survivor curves were derived from plate count data by using a two-phase linear model to determine lag times and slopes of the curves,from which decimal reduction times(D-values)and times to a4-log10inactivation(T4D)were calculated.In media of pH6,the bacteria grew in the presence of#6%NaCl at19and378C and in the presence of#7%NaCl at288C.In media of pH5,growth was observed in the presence of#2,#4,#4,and0.5%NaCl at37,28,19,and128C,respectively.Growth did not occur and bacterial populations gradually declined in media of pH4.While NaCl had a major effect on growth,bacterial survival was affected to a lesser https://www.sodocs.net/doc/357476885.html,g times decreased with increasing NaCl levels;however,the effect on D-values and T4D values was less pronounced.

The average T4D values for media of pH4containing0.5to6%NaCl were4,13,23,and61days at37,28,19,and128C, respectively.These results show that S. exneri is salt tolerant and suggest that salty foods may serve as vehicles for infection with this bacterium.

Shigella,a major cause of foodborne gastrointestinal illness(13),is a highly host-adapted pathogen infecting only humans and certain other primates.The incidence of Shigella infection in the United States has been estimated by the Centers for Disease Control and Prevention at 448,000cases per year,with20%of these cases being due to foodborne transmission of the pathogen(9).Epidemio-logical studies indicate that fecally contaminated water and infected food handlers with poor personal hygiene are fre-quently associated with shigellosis outbreaks.Foods in-criminated in shigellosis outbreaks are generally foods that are consumed raw,multiple-ingredient foods(e.g.,salads), and cooked foods that are not reheated before serving.Sev-eral outbreaks of Shigella sonnei infection associated with the consumption of chopped uncooked curly parsley oc-curred in the United States and Canada in July and August of1998.Investigations implicated parsley imported from a farm in Mexico as the source of these outbreaks(2).More recently,a multistate outbreak of S.sonnei infection was associated with the consumption of a nationally distributed dip consisting of layers of beans,salsa,guacamole,nacho cheese,and sour cream(3).

To develop strategies to control this bacterium in food, it is necessary to evaluate its potential for growth and sur-*Author for correspondence.Tel:215-233-6655;Fax:215-233-6581; E-mail:lzaika@https://www.sodocs.net/doc/357476885.html,.

?Mention of brand or rm names does not constitute an endorsement by the U.S.Department of Agriculture over others of a similar nature not mentioned.

?Presented in part at the87th Annual Meeting of the International As-sociation for Food Protection in Atlanta,Georgia,6to9August2000.vival as a function of nutritional factors and environmental conditions.Shigella spp.survive for extended periods in various foods,even in salty foods(12,15).Survival of Shigella in autoclaved or ltered seawater for.69days at 158C has been reported(10).However,little if any quan-titative data are available,since many of the published re-ports do not provide suf cient experimental details.A re-view of published reports on the growth and survival of Shigella spp.in media and in foods has recently been pub-lished(8).

We previously determined the growth kinetics values for Shigella exneri5348in brain heart infusion(BHI) broth as a function of temperature,pH,and NaCl and NaNO2concentrations,and we developed models to predict the growth of the organism under aerobic(19)and anaer-obic conditions(18).These studies indicated that growth inhibition increased with increasing sodium chloride con-centration in combination with decreasing temperature and decreasing pH.We also determined the survival character-istics of S. exneri5348in low-salt media(0.5%NaCl) with pHs of2to5at temperatures of4to378C(16).

The objective of the present work was to systematically study the effect of increasing NaCl concentrations on the survival of S. exneri in a microbiological medium under conditions that do not support growth.The experimental conditions chosen for the study were within the range en-countered in the preparation and storage of various salt-containing foods such as salads(potato salad,meat salad, sh salad),cured or pickled foods,sausages,cheeses,and ready-to-eat foods.The salt content of these foods is gen-

J.Food Prot.,Vol.65,No.5EFFECT OF NACL ON SHIGELLA

775

FIGURE 1.Effect of NaCl (5,6,7,and 8%)on S. exneri 5348at 288C in BHI broth adjusted to pH 6.

FIGURE 2.Effect of NaCl (0.5,2,4,and 6%)on S. exneri 5348at 378C in BHI broth adjusted to pH 5.

TABLE 1.Effect of NaCl concentration and pH on S. exneri in BHI broth at 378C a

pH NaCl (%)

n

t L (h)

D -value (h)

T 4D (h)

4

5

0.52460.52222131.4(8.5)30.4(15.3)17.0(2.1)0.0(0.0)Growth 8.7(1.2)14.6(2.1)21.7(0.2)25.6(3.0)66.2(13.2)88.7(7.0)103.6(1.3)102.5(11.9)

6

2466811124Growth 0.0(0.0)0.0(0.0)

Decline,then growth

0.0(0.0)

52.758.822.7(1.5)211.0235.4

90.8(5.9)

a

n,number of replicate cultures;t L ,duration of the lag period prior to the commencement of inactivation.Values in parenthe-ses are standard deviations.

erally in the range of 1to 6%(14).The data obtained will be incorporated into the development of a nonthermal in-activation model.

MATERIALS AND METHODS

Microorganism.S. exneri 5348(obtained from Dr.David W.Niesel,University of Texas Medical Branch,Galveston,Tex.)was used throughout this study.This strain has been studied extensively in our laboratory and was used for the development of growth mod-els for S. exneri (18,19).A stock culture of strain 5348was stored at 2708C in cryovials (Nalgene)containing BHI broth (Difco Lab-oratories,Detroit,Mich.)supplemented with 10%(vol/vol)glyc-erol.To prepare the inoculum for each trial,a 0.1-ml portion of the thawed stock culture was added to 10ml of BHI broth and incu-bated without shaking for 18to 24h at 378C.The resulting sta-tionary-phase cells were used to inoculate test media.

Medium.The BHI broth was supplemented with NaCl to give nal concentrations of 0.5,2,4,5,6,7,and 8%NaCl (wt/vol)adjusted to pH 4,5,and 6with 6N HCl.Portions (100ml)were distributed into 250-ml Erlenmeyer asks capped with foam plugs and sterilized by autoclaving.Autoclaving of the media did not result in signi cant changes in pH (60.1pH unit).An Orion Research ionanalyzer/501(Orion Research,Inc.,Boston,Mass.)equipped with a sealed combination electrode with silver/silver chloride reference (pH 0to 14;VWR Scienti c,San Francisco,Calif.)was used for pH determination.

Culture conditions.Portions (1ml)of the inoculum were added to BHI broth to give an initial population level of 106to 107CFU/ml.The media were incubated on a rotary shaker (150rpm)at the desired temperature (12,19,28,or 378C)until bac-terial populations reached levels undetectable by direct plating (,1.3log 10CFU/ml).With one exception,a minimum of two trials (i.e.,fresh cells)were conducted for each variable combi-nation and treatment,with one to three replicates or asks per treatment per trial.

Determination of microbial populations.Bacterial popu-lation levels were determined at appropriate time intervals by sur-face plating cultures,or dilutions thereof prepared in 0.1%pep-tone water,onto duplicate plates of tryptose agar (Difco)with a Spiral Plater (Model D;Spiral System Instruments,Bethesda,Md.).The plates were incubated for 48h at 378C,and the colonies were counted.

Curve tting.Survivor curves were generated by the method of Buchanan et al.(1), tting the population-versus -time data to the linear function that allows for the presence of a lag period before the beginning of the exponential decline in bacterial pop-ulation density.The model for the survivor curves (the two-phase linear inactivation model)is as follows:

N 5N [t ,t ]0

L N 5N 1s (t 2t )

[t $t ]

(1)

0L L where N is the log 10count of bacteria at time t (log 10CFU/ml),N 0is the log 10count of bacteria at time t 50(log 10CFU/ml),s is the slope of the survivor curve (log 10CFU/ml/h),t is the time (h),and t L is the duration of the lag period (h)prior to the com-mencement of inactivation.The curves were tted using ABA-CUS,a nonlinear regression program that employs a Gauss-New-ton iterative procedure (4).A population decrease of $3log 10CFU/ml was deemed acceptable to generate valid survivor curves.

RESULTS AND DISCUSSION

Our previous studies (17,19)showed that S. exneri 5348can grow readily in media containing up to 5%NaCl.

J.Food Prot.,Vol.65,No.5

776

ZAIKA FIGURE 3.Effect of NaCl (0.5,2,4,and 6%)on S. exneri 5348at 198C in BHI broth adjusted to pH 5.

FIGURE 5.Survival curve obtained with a two-phase model for S. exneri 5348at 128C in BHI broth adjusted to pH 4and con-taining 6%NaCl.

FIGURE 4.Effect of NaCl (0.5,2,4,and 6%)on S. exneri 5348at 128C in BHI broth adjusted to pH 5.

FIGURE 6.Survival curve obtained with a two-phase model for S. exneri 5348at 288C in BHI broth adjusted to pH 4and con-taining 6%NaCl.

This strain grew in BHI broth adjusted to pH 6in the pres-ence of 5,5,4,and 0.5%NaCl at 37,28,19,and 128C,respectively.In the present study,experiments were con-ducted to determine NaCl concentrations that limited the growth of strain 5348.The response of the bacterium to increased levels of NaCl (5to 8%)in BHI broth adjusted to pH 6was determined for incubation at 19,28,and 378C.The maximum NaCl concentration permitting growth at 288C was 7%(Fig.1).The bacteria grew without delay in the presence of 5%NaCl and after a slight initial decrease in population in the presence of 6%NaCl.In media con-taining 7%NaCl,growth occurred after ca.170h of in-cubation following a considerable decrease in population (3log 10CFU/ml).This nding suggests that S. exneri may not survive if the initial population is low,even though it may be capable of growth at a given NaCl level (in this case,7%).Also,for cultures in which growth subsequently occurred (6and 7%NaCl),the rate of population decline was greater than that for cultures containing 8%NaCl,in which growth did not occur (Fig.1).At 19and 378C (data not shown),growth was observed in the presence of 6%NaCl but not in the presence of 7%NaCl.At the higher

NaCl concentrations that permitted growth,bacterial pop-ulations initially declined before growth was initiated,and the rate of decrease was the same as or greater than that at NaCl levels that did not support growth.

In media adjusted to pH 5,S. exneri grew at 378C in the presence of 2%NaCl but not in the presence of 4%NaCl (Fig.2),while at 198C (Fig.3)and 288C (data not shown),growth was observed in the presence of 4%NaCl after a slight initial decline in the population level.The bacteria grew at 128C in media adjusted to pH 5only at the lowest NaCl concentration tested (0.5%)but survived at the higher NaCl concentrations for up to 1,000h (Fig.4).The possibility that the organism may be capable of growth in the presence of 2and 4%NaCl is suggested by the initial decline in the population level (ca.1log 10CFU/ml)followed by stable population levels.The bacterial pop-ulation level was essentially unchanged in the presence of 6%NaCl for the duration of the experiment (Fig.4).Pre-viously we observed that with an initial S. exneri 5348

J.Food Prot.,Vol.65,No.5EFFECT OF NACL ON SHIGELLA

777

TABLE 2.Effect of NaCl concentration and pH on S. exneri in BHI broth at 288C a

pH

NaCl (%)

n

t L (h)

D -value (h)

T 4D (h)

4

5

0.52460.544422116.3(5.1)144.5(45.4)129.2(42.4)0.0(0.0)Growth 37.6(13.1)45.6(12.4)47.6(11.6)87.3(2.5)266.8(56.2)326.8(5.9)319.1(8.9)349.1(10.2)

6

246868226444Growth

Decline,then growth

0.0(0.0)0.0(0.0)

Decline,then growth

27.0(10.7)

86.2(26.4)72.2(25.1)128.5(16.2)

309.3(167.2)288.6(100.6)541.2(60.6)

a

n,number of replicate cultures;t L ,duration of the lag period prior to the commencement of inactivation.Values in parentheses are standard deviations.

TABLE 3.Effect of NaCl concentration and pH on S. exneri in BHI broth at 198C a

pH

NaCl (%)

n

t L (h)

D -value (h)

T 4D (h)

4

0.5246856564267.5(69.6)310.4(170.8)128.5(117.3)29.9(46.4)0.0(0.0)50.3(21.7)106.2(82.8)106.8(68.5)99.6(76.1)18.8(9.9)468.9(151.7)735.3(397.3)555.8(375.0)428.4(350.5)75.1(39.4)

5

0.52462223Growth Growth

Decline,then growth

0.0(0.0)192.2(27.6)766.1(106.0)6

846832430.0(0.0)Growth

Decline,then growth

0.0(0.0)

123.0(47.4)267.4(127.9)491.9(189.7)

1,069.6(511.6)

a

n,number of replicate cultures;t L ,duration of the lag period prior tot he commencement of inactivation.Values in parentheses are standard deviations.

population of 3log 10CFU/ml,growth was obtained at 198C in BHI broth adjusted to pH 5in the presence of 0.5%NaCl but not in the presence of 2.5%NaCl,while growth did not occur at 128C (17).Thus,aside from the cultural and en-vironmental conditions,the likelihood of growth may de-pend on the initial population of bacteria.S. exneri 5348did not grow in BHI broth adjusted to pH 4for all NaCl levels tested,while bacterial survival increased with de-creasing temperature (data not shown).

The two-phase linear model of Buchanan et al.(1)was used to t the bacterial population data (equation 1)and gave a convenient mathematical representation of S. exneri inactivation under the nongrowth conditions studied.Figure 5shows a typical survivor curve consisting of a lag period and a declining curve.Figure 6shows a survivor curve without a measurable lag time,which is characteristic of survival in the presence of high NaCl levels ($6%),par-ticularly at the higher temperatures studied (28and 378C).

A summary of the inactivation parameters (lag times,decimal reduction times [D -value],and times to a 4-log 10inactivation [T 4D ])derived from the individual survivor

curves for selected variable combinations of pH and NaCl concentrations at 37,28,19,and 128C are presented in Ta-bles 1through 4,respectively.For some variable combi-nations,relatively large standard deviation values were ob-tained for the inactivation kinetics parameters,particularly at 198C (Table 3).These large values were mainly due to variations in the rates of bacterial inactivation obtained in different trials.Replicates within a trial generally showed good agreement.The time to a 4-log 10(99.99%)inactiva-tion (T 4D 5t L 1[43D ]),which includes both the lag time and the population decline,was a convenient way to compare the response of S. exneri under various combi-nations of temperature,pH,and NaCl concentration.In gen-eral,T 4D values increased with increasing pH and decreas-ing temperature.Increasing concentrations of NaCl led to a decrease in lag times (t L ),while D -values (calculated by taking the negative reciprocal of the s term in equation 1)and T 4D values were affected to a lesser extent.Although NaCl had a major inhibitory effect on S. exneri growth (17),it exerted a lesser effect on the organism’s ability to survive,even in the presence of 8%NaCl.

J.Food Prot.,Vol.65,No.5 778ZAIKA

TABLE4.Effect of NaCl concentration and pH on S. exneri in BHI broth at128C a

pH NaCl

(%)n

t L

(h)

D-value

(h)

T4D

(h)

40.5

2

4

62

2

2

2

701.8(6.6)

1,032.6(5.6)

231.4(110.0)

532.8(257.6)

172.1(23.7)

186.4(42.4)

224.5(4.3)

266.1(37.1)

1,390.4(101.6)

1,778.1(163.9)

1,129.5(92.9)

1,597.2(109.1)

50.5

2

4

62

2

2

2

Growth

Decline of1log in1,000h

Decline of1log in1,000h

No change in1,000h

a n,number of replicate cultures;t L,duration of the lag period prior to the commencement of inactivation.Values in parentheses are standard deviations.

The survival of21strains each of S. exneri and S. sonnei at room temperature in peptone water was studied by Fehlhaber(6).All strains survived for at least4days in the presence of6%NaCl,while three strains of S.sonnei and one strain of S. exneri survived for21days.Our re-sults suggest that S. exneri is resistant to the action of NaCl,particularly at198C(Table3)and128C(Table4). However,in contrast to our study,Fehlhaber employed cells that were apparently in the mid-exponential phase of growth(i.e.,5-h cultures at378C in nutrient broth).It is generally recognized that bacterial resistance to stress fac-tors is stronger for cells in the stationary phase than for those in the exponential phase of growth(7).

Our own work(17)and that of other investigators(12,

15)indicates that Shigella are able to grow and survive in

a variety of foods;however,the in uence of salt on survival has not been extensively studied.Salads are generally foods of low pH and contain salt.Ra i and Lunsford(11)studied the survival of an avirulent mutant strain of S. exneri5 (pHS1059)inoculated into commercially prepared salads. The salt content was not given,but the pH range of the carrot salad was2.7to2.9,that of the potato salad was3.3 to4.4,that of the coleslaw was4.1to4.2,and that of the cra

b salad was4.4to4.5.The organism survived in all of the salads at48C for at least11days and in crab salad for up to20days.According to Siegmund(12),the average survival times for strains of S. exneri and S.sonnei at228C in sauerkraut(pH3.9),in herring brine containing25% NaCl(pH4.1),and in meat salad(pH5.0)were16,3,and 32days,respectively,and at48C the survival times were 22,21,and52days,respectively.

In conclusion,a study of the survival characteristics of S. exneri as affected by NaCl(0.5to8%)was conducted using BHI broth adjusted to pH4,5,and6at12,19,28, and378C.The results of the study indicate that the organ-ism can tolerate high levels of NaCl,such as those that may be encountered in foods such as pickled vegetables,caviar, pickled herring,dry cured ham,and certain cheeses.These data also reveal that Shigella are capable of surviving for long periods(2weeks to2months),particularly at tem-peratures of,198C.Considering that the infective dose of Shigella may be as low as10to500organisms(5),this nding suggests that foods with pHs of#5,even those containing moderate quantities(2to4%)of salt,stored at or below room temperature may permit the survival of the organism over long periods in suf cient numbers to cause gastrointestinal illness.The survival data obtained in this study will be incorporated into the development of a non-thermal inactivation model that can be used to predict in-activation kinetics parameters such as lag time,D-value, and T4D for S. exneri as a function of temperature,pH, NaCl,and additional factors,such as preservatives,impor-tant to its survival in foods.

ACKNOWLEDGMENT

I thank Marangeli Osoria for skilled technical assistance in conducting the bacterial survival studies.

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一般过去式 时间状语:yesterday just now (刚刚) the day before three days ag0 a week ago in 1880 last month last year 1. I was in the classroom yesterday. I was not in the classroom yesterday. Were you in the classroom yesterday. 2. They went to see the film the day before. Did they go to see the film the day before. They did go to see the film the day before. 3. The man beat his wife yesterday. The man didn’t beat his wife yesterday. 4. I was a high student three years ago. 5. She became a teacher in 2009. 6. They began to study english a week ago 7. My mother brought a book from Canada last year. 8.My parents build a house to me four years ago . 9.He was husband ago. She was a cooker last mouth. My father was in the Xinjiang half a year ago. 10.My grandfather was a famer six years ago. 11.He burned in 1991

学生造句--Unit 1

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base on的例句

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翻译加造句

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