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JFS M: Food Microbiology and Safety

Acid Resistance of Twelve Strains ofEnterobacter sakazakii, and the Impact ofHabituating the Cells to an Acidic EnvironmentS. EDELSON-MAMMEL, M.K. PORTEOUS, AND R.L. BUCHANAN

ABSTRACT: The association of powdered infant formula with cases of severe Enterobacter sakazakii infections inimmunocompromised and premature neonates has led to a need to learn about the basic behavior of this emergingpathogen in food systems and the environment. The current study examines the microorganism’s stationary-phaseacid resistance using 12 strains that had been previously used to characterize its thermal resistance. Acid resistancewas determined by initially culturing the isolates for 18 h in brain heart infusion broth (BHI) at 36 ◦C, transferringthe cells to tryptic soy broth (TSB) adjusted to pH 3.0 and 3.5, and determining E. sakazakii survival over the courseof 5 h incubation at 36 ◦C. At pH 3.5, 10 of the 12 strains showed less than a 1 log cycle decline over the 5-h incubationperiod, with the most acid sensitive strain showing an approximate 3.5 log cycle decline. At pH 3.0, the declineover the 5-h incubation period ranged from 4.9 to >6.3 log cycles; however, substantial diversity was evident whenthe 1-h/pH 3.0 results were compared. The effect of habituating the cells to a moderately acidic environment wasdetermined by growing the strains in TSB with 0% (nonacidogenic) and 1% glucose (acidogenic), transferring thecells to acidified (pH 3.0) BHI, and determining E. sakazakii survival over the course of 5 h of incubation at 36 ◦C.While there was diversity observed among the strains, in general the stationary-phase acid resistances of severalof the strains were enhanced, at least transitorily, by growth in the acidogenic medium. No apparent correlationbetween the stationary-phase relative acid resistances of the strains based on the 1-h/pH 3.0 acid inactivation valuesand the previously reported thermal D-values was observed.

Keywords: acid tolerance, pH, stationary phase

Introduction

E nterobacter sakazakii is an opportunistic pathogen that hasbeen associated with rare, but life-threatening infections (for

example, meningitis, septicemia, necrotizing colitis) in infants, par-ticularly premature neonates and infants with underlying chronicconditions (FAO/WHO 2004, 2006). In several incidences the sourceof the microorganisms has been linked to dehydrated powderedinfant formula (Arseni and others 1987; Biering and others 1989;Simmons and others 1989; Noriega and others 1990; Van Acker andothers 2001; Himelright and others 2002). Surveys have detected themicroorganism in powdered infant formulas at frequencies rangingfrom 0% to 22%, but the microorganism is typically present at levelsbelow 1 CFU/100 g of dry powder (FAO/WHO 2006). It is presumedthat most cases involve outgrowth of E. sakazakii after rehydrationof the formula.

While this microorganism does appear to be a relatively com-mon minor constituent of the microflora of various foods and foodmanufacturing environments (Kandhal and others 2004), there isrelatively little information available about its growth and survivalcharacteristics. The availability of such knowledge is critical to thedevelopment of methods for preventing, controlling, or eliminatingthis microorganism from powdered infant formula and the areasin which the product is manufactured, prepared, and consumed.

MS 20060080 Submitted 2/5/2006 Accepted 5/19/2006. Authors Edelson-Mammel and Buchanan are with DHHS Food and Drug Administration,Center for Food Safety and Applied Nutrition, College Park, Md., U.S.A. Au-thor Porteous is with Univ. of Maryland, College Park, Md., U.S.A. Directinquiries to author Buchanan (E-mail: robert.buchanan@fda.hhs.gov).

In response to the emergence of this enteric microorganism as apublic health concern, there has been a concerted effort during thepast several years by several laboratories worldwide to learn moreabout the microorganism’s growth and survival characteristics infood systems, including establishing the genotypic and phenotypicvariability among strains of the species. To date, the characteristicsstudied most intently have been the microorganism’s thermal resis-tance (Kindle and others 1996; Narzarowec-White and others 1999;Breeuwer and others 2003; Edelson-Mammel and Buchanan 2004;Iversen and others 2004; Williams and others 2005) and its ability tosurvive dehydration (Breeuwer and others 2003; Edelson-Mammeland others 2005).

One of the basic characteristics of foodborne pathogens that in-fluence their behavior in foods is the ability to tolerate acidic or al-kaline conditions. The ability to tolerate short- and long-term expo-sures to acidic or alkaline pH environments influences their survivalin food, their susceptibility to certain control strategies, and theirrelative virulence (Smith 2003). While it has been presumed that E.sakazakii tolerates acidic pH exposures in a manner similar to thatobserved with other Enterobacteriaceae, there are currently few dataavailable on its acid resistance. Richards and others (2005) reportedthat infant rice cereal rehydrated with apple juice (final pH = 4.39)did not support the growth of E. sakazakii. Kim and Beuchat (2005)observed that E. sakazakii did not grow in apple (pH 3.9) and straw-berry (pH 3.6) juices stored at 25 ◦C, but did grow in tomato (pH 4.4),watermelon (pH 5.0), and cantaloupe (pH 6.8) juices. However, todate there appears to be no systematic evaluation of the microorgan-ism’s acid resistance or the potential for augmenting that resistancethrough inducible pH-dependent, stationary-phase acid resistance.

No claim to original US government worksC© 2006 Institute of Food Technologists Vol. 71, Nr. 6, 2006—JOURNAL OF FOOD SCIENCE M201doi: 10.1111/j.1750-3841.2006.00101.xFurther reproduction without permission is prohibited

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Accordingly, the goals of the current study were to determine the in-herent pH resistance of stationary-phase cells of E. sakazakii, andto determine if that resistance can be increased by acid habituation.The study examined a total of 12 strains from clinical, environmen-tal, and food sources to evaluate the biological diversity of the mi-croorganism’s response to an acid stress. The strains selected werethe same as those used in an earlier studies of thermal resistance(Edelson-Mammel and Buchanan 2004; Williams and others 2005)to determine if there is any correlation between acid resistance andthermal resistance among the strains.

Materials and Methods

MicroorganismsThe same 12 strains (4.01C, 607, 29544, 51329, NQ1-Environ,

NQ2-Environ, NQ3-Environ, LCDC648, LCDC 674, CDC A3(10),SK90, EWFAKRC11NNV1493) employed in our earlier investiga-tion of the thermal resistance of E. sakazakii (Edelson-Mammeland Buchanan 2004) were used in the current study to determinethe stationary-phase acid resistance. The sources of the culturesand how they were maintained have been described previously(Edelson-Mammel and Buchanan 2004).

Determination of stationary-phase acid resistancePreparation of inocula. The 12 strains were individually grown

to stationary phase by inoculating sterile test tubes containing 10 mLof brain heart infusion broth (BHI) (Becton Dickinson, Sparks, Md.,U.S.A.) and incubating the cultures without agitation for 18 h at36 ◦C. The level of E. sakazakii in the 18-h BHI cultures was typicallybetween 108 and 109 CFU/mL.

Assessing acid resistancePortions of tryptic soy broth (TSB) were prepared according to

the manufacturer’s directions and sterilized by autoclaving. The pHof the broth was then adjusted to either pH 3.0 or pH 3.5 with 10N HCl and resterilized by filtration (0.2 μm). Hydrochloric acid wasselected to acidify the TSB so that the effect of pH was evaluatedwithout confounding anion effects that occur with weak organicacids. The pH-adjusted TSB was then aseptically dispensed in 10-mL portions into sterile culture tubes. The TSB-containing culturetubes were preequilibrated to 36 ◦C prior to the transfer to avoidthermal shock. Each of a set of 18 culture tubes for each strain/pHcombination was then inoculated with 0.1 mL of the appropriate18-h BHI culture (see above) and placed in a 36 ◦C incubator. Theinitial level of E. sakazakii in these TSB tubes was approximately 107

CFU/mL.At 0, 1, 2, 3, 4, and 5 h, 3 tubes for each strain/pH combination

were removed from the incubator and appropriate dilutions madeusing sterile 0.1% peptone water. The samples and dilutions werethen plated onto duplicate tryptic soy agar (TSA) plates using a spiralplater (Autoplater 4000, Spiral Biotech, Bethesda, Md., U.S.A.). Theplates were incubated for 18 to 20 h at 36 ◦C, enumerated with theautomatic laser plate counter (Spiral Biotech), and transformed tolog (CFU/mL) values. In those instances where no colonies weredetected, a log (CFU/mL) value of 0.70 was assigned, this being halfof the lower limit of detection for the assay.

Evaluation of the strains for inducible pH-dependent,stationary-phase acid resistance

Preparation of inocula. The dual medium system of Buchananand Edelson (1996) was used to induce or not induce pH-dependent

stationary-phase acid resistance. Tryptic soy broth without dex-trose (TSB-G) was prepared as per the manufacturer’s instructionsand divided into 2 portions. One portion (TSB+G) was supple-mented with glucose to a level of 1% glucose. The 2 media weredispensed in 10-mL portions to capped culture tubes and thenautoclaved. Each of the strains was individually inoculated into1 TSB+G and 1 TSB-G culture tube, with all tubes then beingincubated for 18 h at 36 ◦C. After 18 h, the cultures were wellinto the stationary phase, having counts of approximately 108 and109 CFU/mL.

Characterization of pH changes during culturing of 18-h cul-tures. The effectiveness of the dual medium system to yield culturesthat were or were not habituated to an acidic pH was verified in aseries of preliminary experiments. Sets of 15 culture tubes contain-ing 10 mL of TSB-G or TSB+G were prepared and sterilized. Eachtube in the set was then inoculated with 0.1 mL of an 18-h BHIculture of one of the E. sakazakii strain (see above) and the tubeswere incubated at 36 ◦C without agitation. At designated times atube was removed and the pH was determined using a combinationelectrode and pH meter (model 8005, VWR Scientific, West Chester,Pa., U.S.A.). This procedure was repeated on 6 different occasionsfor each strain/medium combination.

Assessing inducible pH-dependent, stationary-phase acid re-sistance. Acid resistance was evaluated using a modification of thesystem described above. BHI was prepared, sterilized by autoclav-ing, and the pH adjusted to 3.0 using 1 N HCl. After resterilizationby filtration (0.2 μm), the BHI was aseptically dispensed in 10-mLaliquots into sterile culture tubes. The tubes were then equilibratedat 36 ◦C by placing them in an incubator.

A set of 21 pH 3.0 BHI tubes were then inoculated with 0.1 mLof an 18-h TSB-G culture of a single strain of E. sakazakii. A sec-ond set of 21 tubes were inoculated with the TSB+G 18-h culture ofthe same strain. The initial level of E. sakazakii was approximately107 CFU/mL. After 0, 0.5, 1, 2, 3, 4, and 5 h, triplicate culture tubeswere removed and assayed for viable cells by surface plating appro-priate dilutions on duplicate TSA plates using a spiral plater (SpiralBiotech). The plates were incubated at 36 ◦C for 18 to 20 h, enu-merated using automatic laser plate counter, and then transformedto log (CFU/mL) values. Again, when no viable colonies were ob-served, a log (CFU/mL) = 0.70 was assigned. The extent of induciblepH-dependent stationary-phase acid resistance was then assessedby examining the differential in survivor rates between the TSB+Gand TSB-G grown cells.

Results

Stationary-phase acid resistanceThe ability of E. sakazakii to tolerate moderately acidic conditions

was strongly pH dependent; survival was substantially different atpH 3.0 and 3.5. Almost all of the 12 strains were able to tolerate the5-h challenge at pH 3.5 with only small declines in viable counts(Table 1). Ten of the 12 strains showed less than a 0.5 decline in log(CFU/mL) counts. The 2 strains that showed the greatest averagedeclines at pH 3.5, 1.1, and 3.5 log (CFU/mL) were ATCC 29544 andATCC 51329, respectively.

Exposure to pH 3.0 produced a much more extensive and rapidloss of viability (Table 2). All strains decreased by>4 log cycles duringthe 5-h exposure period, with most strains decreasing ≥6 log cycles.Two types of inactivation kinetics were apparent (Figure 1). The moreresistant strains, such as SK90, displayed a log-linear decline overthe course of the exposure period. Other less resistant strains, suchas LCDC 648, displayed very rapid inactivation during the 1st h, fol-lowed by slow decline of the surviving cell population. Strain ATCC

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Acid resistance of E. sakazakii . . .

51329 continued to be the most sensitive strain, but ATCC 29544 sur-vived longer than several of the strains that had been more resistantat pH 3.5 (Table 1).

While all 12 strains were clearly sensitive to a 5-h exposure topH 3.0, when viewed over shorter exposure times, it is apparentthat there was substantial variability among the isolates. For exam-ple, if the extent of inactivation is considered after the 1st h only(Figure 2A), approximately a third of the strains were very sensi-tive to exposure to a pH 3.0 environment. After the 2nd h of expo-sure (Figure 2B) the diversity in extent of survival among the strainswas even broader. There appeared to be no correlation between thesources of the strains (that is, clinical compared with food comparedwith environmental) and their acid resistance.

Inducible pH-dependent, stationary-phase acidresistance

As expected, the glucose in TSB+G is metabolized fermentativelyby E. sakazakii, leading to a depression of pH from neutrality to ap-proximately pH 5.0 to 5.2 during stationary phase (Figure 3). Con-versely, growth of E. sakazakii in TSB-G allowed the microorganismto be continuously cultured to stationary phase in a neutral envi-ronment, though a small decrease in pH from approximately 7.3 to7.0 was observed. There was little variability among the 12 strains.Thus, E. sakazakii cultured in TSB+G for 18 h should have beenfully habituated to a moderately acidic environment, whereas the

Table 1 --- Survival of stationary-phase cells of 12 strains of Enterobacter sakazakii when resuspended in acidifiedTSB (pH 3.5) and held for 5 h at 36 ◦C

Duration of exposure to pH 3.5 (h)

Strain 0 1 2 3 4 5

4.01C 6.90a ± 0.12 6.86 ± 0.02 6.83 ± 0.03 6.74 ± 0.02 6.52 ± 0.06 6.39 ± 0.05607 6.84 ± 0.02 6.85 ± 0.03 6.82 ± 0.05 6.82 ± 0.07 6.69 ± 0.03 6.54 ± 0.02ATCC 29544 6.70 ± 0.06 6.62 ± 0.02 6.50 ± 0.01 6.29 ± 0.02 5.94 ± 0.01 5.59 ± 0.03ATCC 51329 6.80 ± 0.05 6.72 ± 0.03 6.39 ± 0.05 5.19 ± 0.26 4.01 ± 0.07 3.34 ± 0.08NQ1-Environ 7.04 ± 0.04 7.01 ± 0.07 6.93 ± 0.01 6.85 ± 0.03 6.74 ± 0.02 6.61 ± 0.03NQ2-Environ 6.78 ± 0.02 6.78 ± 0.05 6.74 ± 0.03 6.71 ± 0.03 6.69 ± 0.02 6.63 ± 0.02NQ3-Environ 6.88 ± 0.01 6.79 ± 0.08 6.74 ± 0.06 6.72 ± 0.02 6.71 ± 0.04 6.78 ± 0.04LCDC 648 6.73 ± 0.03 6.71 ± 0.04 6.71 ± 0.08 6.58 ± 0.02 6.47 ± 0.02 6.25 ± 0.02LCDC 674 6.61 ± 0.07 6.59 ± 0.01 6.61 ± 0.01 6.60 ± 0.03 6.50 ± 0.01 6.55 ± 0.07CDC A3(10) 6.77 ± 0.18 6.83 ± 0.02 6.71 ± 0.02 6.76 ± 0.04 6.71 ± 0.02 6.62 ± 0.06SK90 6.72 ± 0.12 6.68 ± 0.04 6.63 ± 0.04 6.68 ± 0.11 6.62 ± 0.03 6.63 ± 0.03EWFAKRC 11NNV1493 6.76 ± 0.12 6.69 ± 0.10 6.67 ± 0.04 6.62 ± 0.01 6.50 ± 0.03 6.39 ± 0.04

Values are the means of 3 replicates.aMean ± standard deviation.

Table 2 --- Survival of stationary-phase cells of 12 strains of Enterobacter sakazakii when resuspended in acidifiedTSB (pH 3.0) and held for 5 h at 36 ◦C

Duration of exposure to pH 3.0 (h)

Strain 0 1 2 3 4 5

4.01C 6.85a ± 0.02 5.49 ± 0.15 4.21 ± 0.13 2.72 ± 0.63 2.15 ± 1.24 0.70b

607 6.85 ± 0.05 5.83 ± 0.02 3.86 ± 0.17 1.59 ± 0.36 1.80 ± 1.90 0.90 ± 0.35ATCC 29544 6.64 ± 0.03 4.55 ± 0.08 1.90 ± 2.07 1.37 ± 1.16 0.90 ± 0.35 0.70ATCC 51329 6.81 ± 0.08 2.77 ± 0.07 0.70 0.70 0.70 0.70NQ1-Environ 7.03 ± 0.05 5.10 ± 0.01 2.80 ± 0.12 1.40 ± 0.17 1.00 ± 0.52 0.70NQ2-Environ 6.81 ± 0.06 5.25 ± 0.18 2.96 ± 0.17 1.97 ± 0.58 1.20 ± 0.46 1.87 ± 1.56NQ3-Environ 7.02 ± 0.03 6.28 ± 0.37 3.69 ± 1.03 2.48 ± 1.55 3.27 ± .055 0.70LCDC 648 6.70 ± 0.02 1.62 ± 1.59 1.06 ± 0.62 1.28 ± 1.01 0.70 0.70LCDC 674 6.64 ± 0.07 5.10 ± 0.03 3.11 ± 0.08 1.95 ± 0.05 1.25 ± 0.95 0.70CDC A3(10) 6.91 ± 0.02 6.31 ± 0.04 4.86 ± 0.08 3.22 ± 0.04 2.20 ± 0.09 2.00 ± 0.40SK90 6.67 ± 0.06 5.98 ± 0.02 4.84 ± 0.08 3.91 ± 0.05 2.79 ± 0.04 1.65 ± 0.37EWFAKRC 11NNV1493 6.73 ± 0.17 2.82 ± 1.01 0.90 ± 0.35 0.90 ± 0.35 0.70 0.96 ± 0.45

Values are the mean of 3 replicates.aMean ± standard deviation [log (CFU/mL)].bWhen no colonies were detected, a log (CFU/mL) value of 0.70 was assigned. This represents half of the lower limit of detection [LLD = log (CFU/mL) = 1.0].

TSB-G would be nonhabituated. It is worth noting that TSB+G wasconsistently approximately 0.2 pH units less than TSB-G prior to in-oculation (for example, 7.15 compared with 7.35); however, this didnot appear to have had any significant impact on relative growth ofthe microorganism in the two media.

As before, approximate 6-log declines in viable counts were ob-served over the course of the 5-h exposure to pH 3.0 for all strains,regardless of the medium in which they were initially cultured(Table 3). The impact of habituating E. sakazakii to a moderatelyacidic environment on its subsequent ability to survive an expo-sure to more acidic conditions was transitory and varied amongthe strains. Some strains, for example, CDC-A3(10), showed lit-tle if any difference in the survival profiles of TSB+G and TSB-Ggrown cells (Figure 4A). Conversely, other strains (for example, EW-FAKRC11NNV1493) demonstrated increased resistance across thecourse of the entire survival profile as a result of being initially cul-tured in acidogenic TSB+G (Figure 4B). Prior growth in TSB+G pro-vided transitory enhanced acid resistance against the rapid loss ofviability observed with ATCC 51329, the most sensitive strain, im-mediately after transfer to the acidified BHI (Figure 4C). Strain SK90(Table 3) was unique in that the TSB-G cells displayed increasedsurvival at the end of the 5-h acid challenge, though this differentialwas not observed at the earlier sampling times.

As a way of further considering the diversity of responsesamong the different strains immediately after exposure to an acidic

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environment, the survivor data from Table 3 were used to calculatethe ratios of the differences in counts for the 1st 0.5 and 1 h for TSB-Gand TSB+G grown cells for the 12 strains using the formula:

ARR = (LCt0 − LCtx)TSB−G/(LCt0 − LCtx)TSB+G

where ARR=acid resistance ratio, (LCt0 – LCtx)TSB−G = log (CFU/mL)at time 0 – log (CFU/mL) at time X (0.5 or 1.0 h) for TSB-G grown cells,and (LCt0 – LCtx)TSB+G = log (CFU/mL) at time 0 – log (CFU/mL) attime X (0.5 or 1.0 h) for TSB+G grown cells. A ratio of 1.00 would indi-cate that prior growth in TSB+G provided no protection comparedto TSB-G grown cells. Values >1.00 would indicate that prior growthin TSB+G resulted in enhanced stationary-phase acid resistance.Conversely, values <1.00 would indicate that growth in TSB+G ac-tually decreased acid resistance. The frequency distributions for thecalculated ARR values for the 12 strains are provided for the 0.5 hand 1.0 h exposure intervals (Figure 5).

The acid resistance patterns observed when the strains were ini-tially cultured in BHI and then transferred to acidified TSB weregenerally similar to those observed when the strains were initiallygrown in TSB+G and TSB-G and then transferred to acidified BHI(compare Table 2 and Table 3). While not universal, in a numberof instances the acid resistances observed with the BHI to acidifiedTSB transfers were intermediate to the TSB+G to BHI and TSB-Gvalues. It is possible that the small amount of glucose in BHI (0.2%)was enhancing the acid resistance of some strains of E. sakazakiiupon subsequent transfer to acidified TSB.

Discussion

Despite the fact that there is an extensive body of research on thesurvival of foodborne bacteria in acidic environments, it is dif-

ficult to directly compare the acid resistance of E. sakazakii with thatof other pathogens. This reflects the fact that the ability of a microor-ganism to survive an exposure to an acid environment is dependenton a large number of factors such as pH, acidulant identity, acidu-lant concentration, temperature, water activity, atmosphere, andthe presence of other inhibitory compounds (Buchanan and oth-ers 1994, 1997; Buchanan and Golden 1995; Buchanan and Edelson1999). Investigators have employed an array of pH values, acidulants,incubation temperatures, and water activities, making it difficultto quantitatively compare reported acid resistances. This is furthercomplicated by the diversity of acid resistances observed among

0

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Figure 1 --- Examples of the inactivation kinetics observedwhen Enterobacter sakazakii was resuspended at 36 ◦Cin tryptic soy broth adjusted to pH 3.0. Strain SK90: •Strain LCDC 648: � .

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Figure 2 --- Diversity in the extent of inactivation observedamong the 12 strains of Enterobacter sakazakii after be-ing resuspended in acidified tryptic soy broth (pH 3.0) for1 h (A) and 2 h (B) at 36 ◦C

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Figure 3 --- The changes in pH observed when the 12 Enter-obacter sakazakii strains were grown in tryptic soy brothwith 0% glucose (TSB-G) and 1% glucose (TSB+G) for 24h at 36 ◦C. Values are the average of 3 independent tri-als performed on 2 separate occasions (n = 6). Standarddeviations were between 0.02 and 0.10 of a pH unit.

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strains of a species (Buchanan and Edelson, 1996, 1999). However,based on the available literature and the stationary-phase acid re-sistant data from the current study (Table 1 and 2), E. sakazakii ap-pears to be best classified as a moderately acid resistant member ofthe Enterobacteriaceae. It has less resistance than well-documentedacid resistant pathogens such as enterohemorrhagic Escherichiacoli (Buchanan and Edelson 1996, 1999) and Listeria monocytogenes(Buchanan and Golden 1998). Its acid resistance appears similar tothe acid resistance reported by Tetteh and Beuchat (2003a, 2003b)for Shigella flexneri but less than that reported by Gorden and Small(1993). The acid resistance of E. sakazakii also appears similar to thatof salmonellae (Gorden and Small 1993), whereas it is more resistantthan Vibrio parahaemolyticus (Wong and Wang 2004) or Aeromonashydrophila (Isonhood and others 2002).

There was substantial diversity in acid resistance among the 12strains of E. sakazakii examined, particularly when exposed to astressful pH (that is, 3.0) for a short time period. This is consis-tent with the diversity among strains observed with other food-borne pathogens such as enterohemorrhagic Esch. coli (Buchananand Edelson 1996, 1999), Salmonella typhimurium (Berk and oth-ers 2005), and L. monocytogenes (Francis and O’Beirne 2005). De-spite this diversity, substantial inactivation of E. sakazakii did occuramong all strains when exposed to a pH 3.0 for several hours. How-ever, it is important to note that this occurred at an elevated temper-ature (36 ◦C). Based on the behavior of other foodborne pathogens,it can be anticipated that acid inactivation of E. sakazakii would takelonger if the temperature was reduced to refrigeration temperatures.Kim and Beuchat (2005) observed slower inactivation of a 5 strainmixture of E. sakazakii in strawberry juice (pH 3.5 to 3.7) held at 4 ◦Cthan at 12 and 25 ◦C, but this effect was less evident with apple juice(pH 3.9 to 4.0).

The ability of foodborne pathogens to survive acidic stress is en-hanced as the cells enter stationary phase. Stationary-phase acidresistance of many foodborne pathogens can be enhanced further

Table 3 --- Survival of 12 Enterobacter sakazakii strains initially grown in TSB+G or TSB-G, transferred to acidifiedBHI (pH 3.0), and incubated at 36 ◦C for 5 h

Duration of exposure to pH 3.0 (h)Growth

Strain medium 0.0 0.5 1.0 2.0 3.0 4.0 5.0

4.01C TSB+G 7.56 ± 0.19a 7.28 ± 0.24 6.51 ± 0.15 5.82 ± 0.25 3.87 ± 0.49 1.65 ± 0.65 0.90 ± 0.35TSB−G 7.46 ± 0.19 6.31 ± 0.24 5.52 ± 0.15 3.73 ± 0.25 1.26 ± 0.49 1.42 ± 0.64 0.70b

607 TSB+G 7.19 ± 0.03 6.81 ± 0.06 6.55 ± 0.06 4.28 ± 0.26 2.21 ± 0.49 1.74 ± 0.75 1.60 ± 0.30TSB−G 7.05 ± 0.03 6.62 ± 0.05 6.23 ± 0.22 3.63 ± 0.72 1.67 ± 1.42 0.70 0.70

ATTC 29544 TSB+G 7.35 ± 0.12 6.59 ± 0.17 5.81 ± 0.05 3.97 ± 0.19 1.89 ± 0.26 0.70 0.70TSB−G 7.21 ± 0.08 6.99 ± 0.01 4.09 ± 0.20 1.26 ± 0.97 0.90 ± 0.35 0.70 0.70

ATCC 51329 TSB+G 7.45 ± 0.06 6.08 ± 0.10 3.37 ± 0.34 1.57 ± 1.50 1.57 ± 1.50 1.00 ± 0.35 0.70TSB−G 7.41 ± 0.30 3.36 ± 0.10 3.30 ± 0.00 2.47 ± 0.75 1.57 ± 1.50 0.70 0.70

NQ1-Environ TSB+G 7.17 ± 0.07 6.83 ± 0.04 6.56 ± 0.05 3.42 ± 0.09 1.26 ± 0.24 1.00 ± 0.30 0.70TSB−G 7.23 ± 0.43 6.42 ± 0.11 5.25 ± 0.31 1.95 ± 0.65 1.13 ± 0.75 0.70 0.70

NQ2-Environ TSB+G 7.80 ± 0.03 5.59 ± 0.06 5.55 ± 0.16 3.66 ± 0.56 1.50 ± 0.35 1.27 ± 0.98 0.95 ± 0.43TSB−G 7.74 ± 0.06 3.51 ± 0.13 2.17 ± 0.34 1.00 ± 0.52 0.70 0.70 0.70

NQ3-Environ TSB+G 7.09 ± 0.04 5.70 ± 0.18 5.50 ± 0.11 3.18 ± 0.09 1.71 ± 0.49 1.39 ± 0.74 1.00 ± 0.52TSB−G 7.05 ± 0.01 4.50 ± 0.67 2.30 ± 0.09 1.00 ± 0.52 0.90 ± 0.35 0.70 0.70

LCDC 648 TSB+G 7.41 ± 0.13 7.04 ± 0.17 4.71 ± 1.58 3.38 ± 0.66 1.08 ± 0.66 0.90 ± 0.35 0.70TSB−G 7.54 ± 0.06 7.15 ± 0.47 4.41 ± 1.48 1.37 ± 1.15 0.70 0.90 ± 0.35 0.70

LCDC 674 TSB+G 7.19 ± 0.11 6.87 ± 0.06 3.97 ± 0.08 1.26 ± 0.54 0.70 0.70 0.70TSB−G 7.07 ± 0.11 6.04 ± 0.13 3.35 ± 0.53 0.70 0.70 0.70 0.70

CDC A3(10) TSB+G 7.05 ± 0.01 6.80 ± 0.05 5.98 ± 0.02 2.69 ± 0.23 1.36 ± 0.10 0.70 0.70TSB−G 6.99 ± 0.06 6.08 ± 0.07 5.32 ± 0.10 3.00 ± 0.08 0.70 0.70 0.70

SK90 TSB+G 7.42 ± 0.01 7.12 ± 0.06 6.58 ± 0.02 6.41 ± 0.21 3.92 ± 0.04 1.50 ± 0.17 0.70TSB−G 7.58 ± 0.13 7.10 ± 0.10 6.34 ± 0.20 5.67 ± 0.24 3.69 ± 0.62 1.77 ± 1.85 1.68 ± 0.85

EWFAKC 11NNV1493 TSB+G 7.32 ± 0.20 7.55 ± 0.04 5.80 ± 0.12 4.16 ± 0.23 2.25 ± 1.37 0.90 ± 0.35 0.70TSB−G 7.40 ± 0.43 5.80 ± 0.17 4.09 ± 0.15 0.90 ± 0.35 0.70 0.90 ± 0.35 0.70

aMean ± standard deviation of 3 replicate trials.bWhen no colonies were detected, a log (CFU/mL) value of 0.70 was assigned. This represents half of the lower limit of detection [LLD = log (CFU/mL) = 1.0].

by preexposing the cells to a moderately acidic environment prior toan acid stress (Beales 2004). Termed acid adaptation, acid tolerance,inducible acid resistance, and acid habituation depending on themeans of preexposing the cells to a moderately acidic environment,these adaptation mechanisms have been observed in a number offoodborne pathogens such as L. monocytogenes (Buchanan and oth-ers 1994; Samelis and others 2003), enterohemorrhagic Esch. coli(Buchanan and Edelson 1996; Buchanan and others 1997; Samelisand others 2003), Sal. typhimurium (Gorden and Small 1993; Samelisand others 2003; Berk and others 2005), S. flexneri (Gorden andSmall 1993; Tetteh and Beuchat 2003a), A. hydrophila (Isonhoodand others 2002), and Vibrio vulnificus (Bang and Drake 2005). Thestationary-phase acid resistance of at least some E. sakazakii strainscould be enhanced by prior growth in an acidogenic medium (thatis, TSB+G) (Table 3). In general the extent of this enhanced acidresistance was not as great as that observed in enterohemorrhagicEsch. coli and L. monocytogenes, but would be sufficient to enhancesurvival of a transitory exposure to an acidic treatment such as thosethat might be used to remove bacteria from food contact surfaces.It would be expected that the growth of E. sakazakii in milk or hy-drated infant formula would lead to a depression in pH similar tothat noted in TSB+G (Richards and others 2005). The mechanism(s)of acid resistance in E. sakazakii has not been explored in a system-atic manner and will require future research.

As indicated earlier, the 12 strains examined for their stationary-phase acid resistance in the current study were previously evalu-ated for their thermal resistance (Edelson-Mammel and Buchanan2004). The possibility that the ability of E. sakazakii to survive these2 stresses is in some manner related was explored by comparingthe extent of acid inactivation observed with pH 3.0/1-h samplesin the current study (Table 2) with the D-values from the earlierstudy (Figure 6). Regression analysis (R2 = 0.053) of the paired re-sponses for the 12 strains indicates that acid resistance and ther-mal resistance are independent characteristics. A similar lack of

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correlation was observed when the pH 3.0/1-h acid inactivation datafor TSB+G and TSB-G grown cells (Table 3) were compared with theD-values (data not shown). Whether the induction of pH-dependentstationary-phase acid resistance in E. sakazakii enhances its ther-mal tolerance in a manner similar to that noted for other foodbornepathogens will require future research.

Conclusions

E nterobacter sakazakii is moderately acid resistant enteric bac-terium that can withstand transitory exposure to a pH of 3.0

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Figure 4 --- Acid inactivation kinetics observed withEnterobacter sakazakii strains CDC-A3(10) (A), EW-FAKRC11NNV1493 (B), and ATCC 51329 (C), grown ini-tially in acidogenic TSB+G (closed symbols) and nonaci-dogenic TSB-G (open symbols) and then transferred toacidified BHI (pH 3.0) for 5 h at 36 ◦C

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Figure 5 --- Diversity in acid resistance ratios (see text)among 12 strains of Enterobacter sakazakii initiallygrown in acidogenic TSB+G and nonacidogenic TSB-Gand then transferred to acidified BHI for 30 min and 1 h at36 ◦C

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Figure 6 --- Scatter-plot of the 12 Enterobacter sakaza-kii strains comparing the extent of inactivation [log(CFU/mL)] when exposed to pH 3.0 for 1 h versus theirD-values (Edelson-Mammel and Buchanan 2004)

and withstand exposures to pH 3.5 for > 5 h. There was substan-tial diversity in acid resistance among the 12 strains, particularlywhen exposed to the pH 3.0 stress for less than 2 hours. The acidresistance of some, but not all strains of E. sakazakii is enhanced byprior growth in an acidogenic medium that habituates the cells to apH of 5.0 to 5.2. There is no apparent relationship between the acidresistance of individual strains and their thermal resistance.

AcknowledgmentWe would like to thank Shruti Naik for her technical assistance inthe conduct of this study.

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