Performance of Media for Recovering Stressed Cells of Enterobacter sakazakii as Determined Using Spiral Plating and Ecometric Techniques

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2005, p Vol. 71, No /05/$ doi: /aem Copyright 2005, American Society for Microbiology. All Rights Reserved. Performance of Media for Recovering Stressed Cells of Enterobacter sakazakii as Determined Using Spiral Plating and Ecometric Techniques J. B. Gurtler and L. R. Beuchat* Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, Georgia Received 5 May 2005/Accepted 23 July 2005 A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55 C for 5 min), freezing ( 20 C for 24 h, thawed, frozen again at 20 C for 2 h, thawed), acidic ph (3.54), alkaline ph (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21 C for 31 days). Control and stressed were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed, suspensions of stressed were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed of E. sakazakii. The orders of performance of media for recovering stressed were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive. Enterobacter sakazakii is recognized as an emerging pathogen that has been documented to cause septicemia and meningitis in preterm and full-term infants (16, 23, 32, 38, 45). The bacterium also has been associated with necrotizing enterocolitis in neonates (43), as well as infections in elderly immunocompromised individuals (14, 23). Powdered infant formula (2, 3, 7, 15, 16, 31, 34, 36, 38, 39, 43, 45) and powdered milk (13, 15, 21, 31, 36) have been epidemiologically implicated as sources of the pathogen. The method used by the U.S. Food and Drug Administration (FDA) (42) to detect E. sakazakii in powdered infant formula requires rehydration in sterile distilled water overnight at 36 C, followed by enrichment in Enterobacteriaceae enrichment (EE) broth overnight at 36 C, surface plating and streaking on violet red bile glucose (VRBG) agar, incubation overnight at 36 C, subculturing presumptive-positive colonies on tryptic soy agar (TSA), and incubating plates for 48 to 72 h at 25 C. Yellow-pigmented presumptive-positive E. sakazakii colonies are then subjected to confirmation tests using the API 20E biochemical-identification system, which requires an additional 18 to 24 h. EE broth and VRBG agar contain selective and differential ingredients (oxgall and brilliant green in EE broth and bile salts no. 3 and crystal violet in VRBG agar) that * Corresponding author. Mailing address: Center for Food Safety, University of Georgia, 1109 Experiment St., Griffin, GA Phone: (770) Fax: (770) may prevent resuscitation of injured E. sakazakii, precluding their detection in powdered infant formula and other foods. Several media have been recently developed for detecting E. sakazakii in powdered infant formula. Oh and Kang (35) described a fluorogenic, differential, selective medium, Oh and Kang (OK) agar. A fluorogen, 4-methyl-umbelliferyl -D-glucoside, serves as an indicator of the production of -glucosidase by E. sakazakii. Bile salts no. 3 selects for enteric bacteria, and ferric citrate and sodium thiosulfate differentiate H 2 S- producing Enterobacteriaceae. This fluorogen is also present in a differential, nonselective medium, Leuschner, Baird, Donald, and Cox (LBDC) agar, developed by Leuschner et al. (26) for the presumptive detection of E. sakazakii in infant formula. Iversen et al. (18) developed a differential, selective medium (Druggan-Forsythe-Iversen [DFI] medium) for recovering E. sakazakii in powdered infant formula. A chromogen, 5-bromo- 4-chloro-3-indolyl-,D-glucopyranoside, acts as a differential agent. This moiety is cleaved by -glucosidase, creating bluegreen E. sakazakii colonies. A selective agent, sodium desoxycholate, along with sodium thiosulfate and ferric ammonium citrate, is also present in the medium. Hsing-Chen and Wu (17) developed fecal coliform agar (FCA) to recover stressed fecal coliforms. Bromcresol purple is used as an indicator of ph change caused by fermentation of lactose. Calcium lactate, which precipitates around fecal coliform colonies after reacting with carbon dioxide, and bile salts no. 3 act as differential and selective agents, respectively. 7661

2 7662 GURTLER AND BEUCHAT APPL. ENVIRON. MICROBIOL. R&F Enterobacter sakazakii chromogenic medium was developed by Restiano (37). This medium contains a chromogen that causes E. sakazakii colonies to appear blue-black in color. It also contains unidentified dyes and bile salts as differential and selective inhibitory agents. As promising as these media are for recovering E. sakazakii from powdered infant formula and other foods, their relative suitabilities for supporting resuscitation and colony development by after exposure to various stress environments have not been evaluated. We undertook a study with the objectives of determining and comparing the abilities of eight agar media to resuscitate and support colony development by healthy and heat-, freeze-, acid-, alkaline-, and desiccationstressed of E. sakazakii. Spiral plating and ecometric techniques were used to assess the performance of media. MATERIALS AND METHODS Bacterial strains. Four strains of E. sakazakii were examined for their responses to stress conditions and recovery on one nonselective medium (control), one nonselective differential medium, and six differential, selective media. Strain 4921, isolated from powdered infant formula linked to an outbreak of E. sakazakii infections, has the same plasmid and multilocus enzyme profile as clinical isolates from infected infants (38). Strain Frm-TN was isolated from powdered infant formula implicated in a case of E. sakazakii neonatal infection (16, 45). The pulsed-field gel electrophoresis pattern of this strain is identical to that of clinical isolates from the infected infant. Strains ES132 and were isolated from powdered infant formula manufactured in two separate commercial processing facilities. Preparation of for exposure to stress environments. Stock cultures of all strains were stored in aqueous glycerol (15%) solution at 20 C prior to use in experiments. Cultures were streaked onto TSA (Difco, Becton Dickinson, Sparks, Md.), incubated at 37 C for 24 h, and stored at 4 C. Cells were grown in brain heart infusion (BHI) broth (Difco, Becton Dickinson) (10 ml) at 37 C for 24 h, with loop transfers (ca. 10 l) at 24-h intervals immediately before exposing them to stress conditions. Procedure for stressing. In preliminary experiments, were exposed for various times to stress conditions. The number of survivors was determined by plating samples on TSA before and after treatment. Treatment conditions in the experiments reported here were selected to cause ca. 1-log CFU/ml reductions in viable exposed to heat, freeze, acid, and alkaline stress; conditions selected for desiccation stress caused reductions of 0.4 to 0.9 CFU/g. Unstressed (control) and exposed to heat, freezing, acid, alkaline, and desiccation environments were enumerated on media using spiral plating and ecometric techniques. Heat stress. Nine milliliters of sterile potassium phosphate buffer (0.1 M; ph 6.8) in 16- by 125-mm screw cap test tubes were adjusted at 55 C by immersing the tubes in an Isotemp 228 water bath (Fisher Scientific, Pittsburgh, Pa.). One milliliter of a 24-h BHI broth culture of E. sakazakii was deposited in the buffer, and the suspension was heated for 5 min. Heated suspensions were cooled by placing the tubes under running tap water for 1 min, serially diluted in a sterile 0.1% peptone solution, and surface plated on test media. Freeze stress. Cells were freeze stressed by adding 1 ml of a 24-h BHI broth culture to 9 ml of sterile potassium phosphate buffer (0.1 M; ph 6.8) in 16- by 125-mm screw cap test tubes and freezing it at 20 C for 24 h. Suspensions were thawed at 21 C, frozen again at 20 C for 2 h, thawed at 21 C, serially diluted in 0.1% peptone solution, and surface plated on test media. Acid stress. Cells were acid stressed by adding 1 ml of a 24-h BHI broth culture to 9 ml of potassium phosphate buffer (0.1 M) adjusted to ph 3.54 with 85% lactic acid and held at 21 C for 30 min. The ph of the treated suspension was adjusted to 6.41 by depositing 1 ml in 9 ml of potassium phosphate buffer (0.1 M; ph 6.8). Suspensions were serially diluted in 0.1% peptone solution and surface plated on test media. The phs of the 24-h BHI broth culture, the acidified potassium phosphate buffer after adding 1 ml of culture, and the buffer after adding 1 ml of the treated suspension were measured. Alkaline stress. Alkaline stress of E. sakazakii was achieved by adding 1 ml of a 24-h BHI broth culture to 9 ml of potassium phosphate buffer (0.1 M) adjusted to ph with sodium hydroxide (2 M) and holding it at 21 C for 5 min. The ph of the alkaline suspension of was adjusted to 6.89 by adding 1 ml to 9 ml of potassium phosphate buffer (0.1 M; ph 6.8). Suspensions were serially diluted in 0.1% peptone solution and surface plated on test media. The phs of the 24-h BHI broth culture, the alkaline potassium phosphate buffer after adding 1 ml of culture, and the buffer after adding 1 ml of treated suspension were measured. Desiccation stress. Cells were desiccation stressed by spraying 24-h BHI cultures of E. sakazakii onto the surface of a commercially manufactured powdered infant formula (Enfamil with Iron, Infant Formula, Milk-Based Powder; Mead- Johnson Nutritionals, Evansville, Ind.). Powdered infant formula (100 g) was distributed evenly on the bottom of a sterile 30-cm-diameter stainless-steel mixing bowl. Spray-inoculation of the powdered infant formula was done using a chromatography reagent sprayer (model ; Kontes Glass Company, Vineland, N.J.). The sprayer was held 35 cm above the formula and sprayed at ca. 2 lb/in 2 with nitrogen gas as a carrier. The formula was mixed with a sterile spoon between applications of each of four inocula (0.025 ml; 0.75 s) to give a final inoculum of ca. 0.1 ml of culture per 100 g of formula. Inoculated formula was aseptically mixed, deposited in a sterile 500-ml screw cap bottle, hermetically sealed, shaken for 2 min, analyzed for a population of E. sakazakii (day zero analysis, i.e., within 15 min after inoculation), and stored at 21 C for 31 days before being analyzed again for the number of surviving E. sakazakii organisms. Control (uninoculated) or inoculated powdered infant formula (10 g) was thoroughly mixed with 90 ml sterile 0.1% peptone solution, and 250 l was spiral plated on TSA on day 0; 100- l samples were plated on TSA supplemented with 0.1% sodium pyruvate (TSAP) on day 31. The plates were incubated for 24 h at 37 C before being examined for presumptive colonies of E. sakazakii. The water activity (a w ) of powdered infant formula was measured before and after inoculation on day zero and again after storage for 31 days at 21 C using an AquaLab (Pullman, Wash.) model CX2 water activity measurement device. Recovery of unstressed and stressed. Enumeration media were prepared in an A-S-10 Agar Sterilizer (New Brunswick Scientific Co., Edison, N.J.) and dispensed (14 1 ml) in petri dishes (90-mm diameter) using a PourMatic Media Dispenser (model MP-320; New Brunswick Scientific). Suspensions of control (unstressed) and stressed E. sakazakii were surface plated on one nonselective medium, one nonselective differential medium, and six differential, selective media. TSAP. TSAP served as a nonselective control medium to recover healthy and in various states of physiological and structural debilitation caused by exposure to stress environments. Sodium pyruvate was added to TSA at a concentration of 0.1% (wt/vol) for the purpose of enhancing resuscitation of injured. LBDC agar (26). Nutrient agar (Oxoid Inc., Basingstoke, Hampshire, United Kingdom) was prepared according to the manufacturer s instructions, cooled to 50 C, and supplemented with the fluorogen 4-methyl-umbelliferyl-,D-glucoside (50 mg/liter; Sigma Chemical Co., St. Louis, Mo.) prior to being dispensed into petri dishes. This medium is nonselective but differentiates between -glucosidase producers and nonproducers. OK medium. OK medium (35) was prepared by combining tryptone (20 g; Difco, Becton Dickinson), bile salts no. 3 (1.5 g; Difco, Becton Dickinson), agar (15 g), sodium thiosulfate (1.0 g; Sigma Chemical Co.), and ferric citrate (1.0 g; MP Biomedicals, LLC, Aurora, Ohio) with 1 liter of deionized water. The ingredients were dissolved by heating and autoclaved at 121 C for 15 min. After cooling to 50 C, the medium was supplemented with 4-methyl-umbelliferyl-,Dglucoside (50 mg/liter) before being dispensed into petri dishes. FCA. Fecal coliform agar was developed by Hsing-Chen and Wu (17). Leclerc et al. (24) reported that FCA recovered higher numbers of true fecal coliforms than did violet red bile lactose agar, had higher performance in terms of specificity and sensitivity than violet red bile lactose agar, and supported the development of typical E. sakazakii colonies. Two solutions were separately prepared. Solution A contained tryptone (20 g), bile salts no. 3 (1.5 g), lactose (10 g), yeast extract (5 g), sodium chloride (5 g), and 850 ml of deionized water. All ingredients were dissolved in water at 50 C. Solution B contained calcium lactate (14 g), -glycerophosphate (1 g) (glycerophosphate disodium salt pentahydrate; MP Biomedicals, LLC, Aurora, Ohio), and 150 ml of deionized water. All ingredients were dissolved in water at 50 C. Solution B was slowly poured into solution A and heated with mixing until the mixture became cloudy. The ph of the mixture was adjusted to with 1 N NaOH solution. Bromcresol purple (3 ml of a solution consisting of 1 g of bromcresol purple [Acros; Fisher Scientific] in 100 ml of 20% ethanol) and 15 g of agar were added to the solution. The mixture was boiled for 2 min, cooled to 50 C, and dispensed in petri dishes. DFI medium. DFI medium was developed by Iversen et al. (18) and is manufactured by Oxoid as chromogenic Enterobacter sakazakii agar (DFI formulation). The medium was prepared according to the manufacturer s instructions. VRBG agar. VRBG agar (Oxoid) was prepared according to the manufacturer s instructions.

3 VOL. 71, 2005 PERFORMANCE OF MEDIA FOR RECOVERY OF E. SAKAZAKII 7663 TABLE 1. Populations of control, heat-stressed, freeze-stressed, acid-stressed, and alkaline-stressed of a composite of four strains of Enterobacter sakazakii recovered on TSAP, LBDC, and differential, selective media Recovery medium Control Heat-stressed Population (log CFU/ml) recovered a Freeze-stressed Acid-stressed Alkaline-stressed All stress conditions TSAP 9.23 a 8.54 a 8.08 a 8.45 a 8.75 a 8.51 a LBDC 9.04 b b a b a b 0.12 FCA 8.96 bc bc b c b c 0.55 DFI 8.88 bc c b c b d 0.73 OK 8.85 c c b c b cd 0.66 VRBG 8.79 cd c b c b cd 0.71 EE 8.42 d c b c c e 1.12 a Within each column, mean values that are not followed by the same letter are significantly different (P 0.05). b Within a control or stress condition, reduction in the number of recovered on differential, selective media compared to the number of recovered on TSAP. c Within a composite of all stress conditions, reduction in the number of recovered on differential, selective media compared to the number recovered on TSAP. R c EE agar. EE agar was made by adding agar (15 g/liter) to Enterobacteriaceae enrichment broth Mossel (Difco, Becton Dickinson). RF medium. R&F Enterobacter sakazakii chromogenic plating (RF) medium was prepared and provided by R&F Laboratories, West Chicago, Ill. Spiral plating. Suspensions of control and stressed of E. sakazakii were spiral plated in duplicate on all media except RF medium. Spiral plating was done using an Autoplate 4000 Automated Spiral Plater (Spiral Biotech, Norwood, Mass.); 50 l of suspension was plated using the exponential-deposition phase. Plates were incubated for 24 h at 37 C before the number of E. sakazakii colonies was determined. Ecometric evaluation. With the exception of desiccation-stressed E. sakazakii, suspensions of control and stressed were streaked on TSAP, LBDC, and differential, selective media using a modification of the ecometric technique described by Mossel et al. (29). Desiccation-stressed were not plated using the ecometric technique because the population (3.5 to 4.2 log CFU/g) in the powdered infant formula was less than the ca. 7 log CFU needed for the technique. The external surface of the bottom of each 90-mm-diameter petri dish was marked with a felt pen into quadrants numbered 1 through 4. The loop portion of a sterile disposable 10- l plastic inoculating loop (VWR International, Bridgeport, N.J.) was immersed in a suspension of control or stressed, and the suspension filling the loop was deposited on the surface of the agar in the first quadrant. Sequentially, and without reimmersing the loop in the suspension, five parallel lines were streaked on the surface of the agar in each of the four quadrants, with one additional streak transversing the middle of the plate at the convergence point of the quadrants, creating a total of 21 streaks. The plates were incubated at 37 C and examined for growth after incubation for 24 h and again after 48 h. Scoring of plates consisted of assigning a growth index (GI) score of 0 to 4.2 to each plate. Growth indices were calculated by counting the streaks (out of 21) on each plate that yielded at least one colony (1 CFU) after incubation for 24 and 48 h and multiplying that number by 0.2. Statistical analysis. At least three independent replicate trials were conducted using spiral plating and ecometric plating techniques. Mean separation of values was determined by the least significant difference by Student s t test using general linear models on SAS software version 8.0 (Statistical Analysis Systems Institute, Cary, N.C). Significant differences (P 0.05) in the number of control and the number of exposed to a given stress treatment that formed colonies on various test media were also determined. The same analyses were done to determine significant differences in GI scores calculated for control and stressed in suspensions streaked on media using the ecometric technique. RESULTS AND DISCUSSION Recovery of control (unstressed) and heat-stressed by spiral plating. Data for composite populations of control of all test strains and for heat-stressed of all test strains recovered on each test medium were analyzed for significant differences. TSAP performed better than all other media for recovering control and heat-stressed (Table 1). Overall, LBDC recovered higher numbers of unstressed (control) and heat-stressed than most of the differential, selective media. EE agar recovered significantly (P 0.05) fewer control than all other media except VRBG agar. The five differential, selective media performed similarly in recovering heat-injured. LBDC outperformed all selective media except FCA, which was not different than DFI, OK, VRBG, and EE in recovering heat-stressed. The general order of performance of media for recovering heat-stressed E. sakazakii by spiral plating was TSAP LBDC FCA, OK, DFI, VRBG, EE. It was of interest to calculate the theoretical D 55 C value (the time required at 55 C to inactivate 90% of the ) of E. sakazakii. The theoretical D 55 C value for the composite of all four strains, reduced by an average of 0.69 log CFU/ml within 5 min, was calculated to be 7.25 min. Reductions in populations within 5 min at 55 C were 0.43, 0.55, 1.24, and 1.89 log CFU/ml for strains ES132, 4921, , and FRM-TN, respectively. The corresponding D 55 C values were 11.63, 9.09, 4.03, and 2.65 min. These D 55 C values should be considered only as theoretical, however, because reductions in populations resulting from heat treatment were less than 1.9 log CFU/ml and the were subjected to only one heating time. The results show that there is more than a fourfold difference in heat resistance among the four strains, which is not atypical of the disparity in D values for E. sakazakii reported by others. Edelson-Mammel and Buchanan (10) examined the thermal resistances of 12 strains of E. sakazakii in rehydrated powdered infant formula using a submerged coil method apparatus (5). A 19-fold difference in D 58 C of the 12 strains was reported. D 56 C values ranged from to min, which were considerably higher than the four-strain average D 55 C value of 7.25 min calculated in our study. Pooled D values for five food isolates and five clinical isolates heated at 52, 54, 56, 58, and 60 C in rehydrated powdered infant formula were reported by Nazarowec-White and Farber (33) to be 54.82, 18.57, 9.75, 3.44, and 2.15 min, respectively. Breeuwer et al. (4) performed thermal-inactivation studies using E. sakazakii isolated from powdered infant formula manufacturing plants. Cells in the stationary growth phase had D values considerably lower than those reported by Nazarowec- White and Farber (33). Five strains were determined to have D 54 C values of 0.27 to 0.50 min. Two strains had D 53 C values of 8.3 and 20.2 min, D 54 C values of 6.4 and 7.1 min, and D 56 C values of 1.1 and 2.4 min. The D 54 C values are closer to the pooled D 55 C value of 7.25 min observed in our study. Iversen

4 7664 GURTLER AND BEUCHAT APPL. ENVIRON. MICROBIOL. et al. (19) determined D values for the type strain of E. sakazakii (NCTC 11467), as well as a capsulated strain. Cells were heated in infant formula milk and in tryptic soy broth (TSB). D values of 16.4, 5.1, 2.6, 1.1, and 0.3 min at 54, 56, 58, 60, and 62 C, respectively, were reported for the type strain heated in infant formula milk. The lower D values reported by Breeuwer et al. (4), compared to those reported by others (10, 19, 33), could be due in part to variations in strain, composition of heating media, and methodology. We heated E. sakazakii in potassium phosphate buffer at ph 6.8, which is close to the neutral ph disodium hydrogen phosphate-potassium dihydrogen phosphate buffer used by Breeuwer et al. (4). In other studies, infant formula milk or TSB was used as the heating medium. Rehydrated powdered infant formula contains fat, protein, and carbohydrate, which may protect against thermal inactivation. Recovery of freeze-stressed by spiral plating. Analysis of counts for all four strains of freeze-stressed as a composite revealed that TSAP and LBDC performed significantly better than all other media (Table 1). The general order of performance of media used to recover freeze-stressed by spiral plating was TSAP, LBDC FCA, OK, VRBG, DFI, EE. Recovery of acid-stressed by spiral plating. A comparison of media for performance in recovering a composite of values for the four acid-stressed strains showed that they fell into three distinct categories (Table 1). The order of performance was TSAP LBDC FCA, OK, VRBG, DFI, EE. This order differs only slightly from that for freeze-stressed in that TSAP performed better than LBDC for acidstressed. Mean ph values of BHI cultures after incubation for 24 h, after acidification, and after neutralization for the four strains were 6.14, 3.54, and 6.41, respectively. Edelson-Mammel and Buchanan (12) examined 12 strains of E. sakazakii for resistance to low-ph stress. Cells were inoculated into BHI broth and grown to stationary phase by incubating them at 36 C for 18 h. The cultures were then transferred to TSB adjusted to either ph 3.0 or ph 3.5 with hydrochloric acid and held at 36 C for up to 5 h. There were no reductions in the number of viable E. sakazakii in TSB at ph 3.5 during the first 2hof incubation. However, after 5 h, the population of the type strain (ATCC 29544) was reduced by approximately 1 log CFU/ml. The population of strain ATCC decreased by 2, 3, and 4 log units within 3, 4, and 5 h, respectively. The remaining 10 strains decreased by 0.5 log CFU/ml within 5 h. In our study, stressing E. sakazakii in phosphate buffer at ph 3.5 for 30 min at 21 C resulted in reductions of 0.85, 0.64, 0.68, and 1.46 log CFU/ml for strains ES132, 4921, , and Frm-TN, respectively. This lower tolerance to acid stress, compared to the tolerance of strains studied by Edelson-Mammel and Buchanan (10), may have been influenced by factors such as the age of the, composition of media, and time and temperature of exposure of the to acidic environments. Recovery of alkaline-stressed by spiral plating. Data for alkaline-stressed of a composite of all four strains were analyzed to determine the overall rankings of the performance of media. TSAP and LBDC performed significantly better than selective media in recovering alkaline-stressed of E. sakazakii (Table 1). Significantly fewer alkaline-stressed were recovered on EE than on other media. The mean ph value of a composite of 24-h TSB cultures of four test strains of E. sakazakii was After alkalization and after neutralization, ph values were and 6.89, respectively. The order of performance of media used to recover alkaline-stressed by spiral plating was TSAP, LBDC FCA, VRBG, OK, DFI EE. This order is in general agreement with the order observed for recovery of heat-, freeze-, and acid-stressed. Comparison of media for recovering exposed to a composite of stress conditions by spiral plating. Composite values from spiral plating experiments using heat-, freeze-, acid-, and alkaline-stressed were analyzed to determine significant differences in the number of control and stressed E. sakazakii organisms recovered on each test medium (Table 1). A significantly higher number of stressed were recovered on TSAP than on LBDC or differential, selective media. LBDC recovered a higher number of stressed than the differential, selective media. The general order of performance in recovering stressed was TSAP LBDC FCA OK, VRBG DFI EE. These results demonstrate the inferiority of differential, selective media in recovering injured E. sakazakii organisms, regardless of the stress condition causing the injury. LBDC was superior to the differential, selective media, however, making it an attractive candidate for use in direct plating food samples that may contain stressed or injured of E. sakazakii and a low number of background microflora. The high performance of LBDC in recovering stressed E. sakazakii is attributed in part to the fact that it contains no selective ingredients, whereas all other media except TSAP contain ingredients that provide differential, selective characteristics. Nutrient agar, used as a basal medium in LBDC agar, differs from TSA in that it contains Lab-Lemco powder (a meat extract) and an unspecified peptone as nitrogen sources, whereas TSA contains an enzymatic digest of soybean meal and a pancreatic digest of casein as nitrogen sources. Nutrient agar also contains yeast extract. Enterobacteriaceae enrichment broth and VRBG agar are recommended by the FDA for detecting E. sakazakii in powdered infant formula (42). Results from our study indicate that agar made from EE broth, as well as VRBG agar, inhibits the recovery of injured. One of the major sites of debilitation of caused by thermal assault is the destruction of cellular enzymes that protect against UV radiation and destructive oxygen species (9). We supplemented TSA with 0.1% pyruvate to enhance the resuscitation and growth of that may have been injured as a result of exposure to stress environments. Baird-Parker and Davenport (1) demonstrated that pyruvate enhanced the repair of damaged bacterial that were otherwise inhibited in their ability to resist toxic oxidizing substances that were present as a result of the destruction of catalase. Recovery of acid-stressed Salmonella (25) and freeze- and heat-stressed Escherichia coli O157:H7 (9, 27, 28) is enhanced in media containing pyruvate. We hypothesize that supplementation of LBDC with pyruvate would enhance the recovery of injured E. sakazakii, resulting in a performance level equivalent to that of TSAP. Across all stress conditions and test strains examined in our study, there was only a 0.12-log CFU/ml difference in the number of recovered on LBDC versus TSAP (Table 1).

5 VOL. 71, 2005 PERFORMANCE OF MEDIA FOR RECOVERY OF E. SAKAZAKII 7665 TABLE 2. Populations of composites of four strains of control and heat-, freeze-, acid-, and alkaline-stressed of E. sakazakii recovered on TSAP and composites of LBDC and differential, selective media Stress condition Population (log CFU/ml) recovered a TSAP Differential, selective media c R d Control a 9.23 a b 8.86 a 0.37 Thermal a 8.54 bc 0.69 b 7.91 c Freeze a 8.02 c 1.21 b 7.53 d Acid a 8.45 bc 0.78 b 7.53 d Alkaline a 8.75 b 0.48 b 8.35 b a Mean values in the same column that are not followed by the same letter are significantly different (P 0.05). Mean values in the same row that are not preceded by the same letter are significantly different. b Within TSAP or within composites of differential, selective media, reduction in the number of stressed recovered compared to the number of control recovered. c Includes LBDC agar. d Reduction in the number of control or stressed recovered on differential, selective media compared to the number recovered on TSAP. Compared to TSAP, recovery of control and heat-, freeze-, acid-, and alkaline-stressed E. sakazakii on LBDC was reduced by 0.19, 0.23, 0.06, 0.24, and 0.05 log CFU/ml, respectively. This placed LBDC in a statistically lower grouping than that of TSAP for recovering stressed of E. sakazakii, but the overall performance of LBDC for recovering was superior to those of the five differential, selective media. The addition of sodium pyruvate to the selective media evaluated in our study may also prove beneficial in resuscitating injured E. sakazakii. The high performance of LBDC in recovering E. sakazakii would argue for its use as an alterative to traditional recovery media, particularly for products containing low populations of background microflora. An added advantage of LBDC over TSAP is the ease of detecting fluorescent colonies of E. sakazakii. The OK agar, like LBDC, also contains 4-methyl-umbelliferyl-,D-glucoside as an indicator of the presence of -glucosidase-producing bacteria, such as E. sakazakii. Blue fluorescence of colonies is more pronounced when viewed under a wavelength of 365 nm than under 245 nm. Viewing the surface of agar on individual petri dishes with a UV lamp for detecting presumptive-positive identification of E. sakazakii is more time-consuming than detecting presumptive-positive colonies using a chromogenic medium such as DFI. The behavior of E. sakazakii after exposure to the four stress conditions was further analyzed by comparing the recovery of a composite of all test strains on TSAP and on a composite of LBDC and differential, selective media (Table 2). Significantly higher (P 0.05) numbers of control and stressed were recovered on TSAP than on a composite of LBDC and differential, selective media. Control, spiral plated on TSAP and a composite of LBDC and differential, selective media, were recovered in significantly higher populations (9.23 and 8.86 log CFU/ml, respectively) than stressed that had been exposed to heat, freezing, acid, or alkaline conditions. When stressed were plated on a composite of LBDC and differential, selective media, the order of ability of stressed to recover was alkaline-stressed thermally stressed acid-stressed freeze-stressed. TABLE 3. Water activities of powdered infant formula and populations of of Enterobacter sakazakii recovered from formula initially (day 0) and after storage at 21 C for 31 days Strain a w of formula and population recovered a Day 0 b Day 31 c R d a w log CFU/g a w log CFU/g ES a 4.63 a b 4.17 a a 4.55 b b 3.99 b a 4.33 c b 3.48 c 0.85 Frm-TN a 4.05 d b 3.68 c 0.37 a The a w of powdered infant formula before spray inoculation was The a w values listed for day 0 were measured within 2 h after spray inoculation. Mean values for populations in the same row that are not preceded by the same letter are significantly different. Mean values for populations in the same column that are not followed by the same letter are significantly different (P 0.05). b Recovery on TSA. c Recovery on TSAP. d Reduction in the number of E. sakazakii in powdered infant formula during storage for 31 days at 21 C. Recovery of desiccation-stressed by spiral plating. Reductions of E. sakazakii populations in powdered infant formula stored for 31 days at 21 C are shown in Table 3. Decreases in populations of the four strains recovered on nonselective media ranged from 0.37 to 0.85 log CFU/g. Strain 4921 was not recovered on EE (minimum detection limit, 50 CFU/g of formula). Composite populations of all four strains of desiccationstressed recovered were analyzed to determine the performance of TSAP, LBDC, and differential, selective media in resuscitating and supporting colony formation. The order of performance of the media was TSAP, LBDC, FCA, OK DFI, VRBG, EE (Table 4). This order is similar to that observed for E. sakazakii exposed to other stress conditions in that TSAP and LBDC performed better than differential, selective media. The results further support the use of LBDC as a direct-plating medium for detecting injured E. sakazakii in powdered infant formula containing a low number of background microflora. Other researchers have investigated the response of E. sakazakii to desiccation stress. Edelson-Mammel and Buchanan (11) inoculated E. sakazakii into powdered infant formula in a TABLE 4. Populations of desiccation-stressed of a composite of four strains of Enterobacter sakazakii recovered from infant formula stored at 21 C for 31 days on TSAP, LBDC, and differential, selective media Recovery medium Population (log CFU/g) recovered a TSAP 3.91 a LBDC 3.92 a 0.01 FCA 3.96 a 0.05 DFI 3.63 b 0.28 OK 3.87 a 0.04 VRBG 3.57 b 0.34 EE 3.44 b 0.47 a Mean values that are not followed by the same letter are significantly different (P 0.05). b Reduction (or increase, ) in the number of desiccation-stressed recovered on differential, selective media compared to the number recovered on TSAP.

6 7666 GURTLER AND BEUCHAT APPL. ENVIRON. MICROBIOL. Recovery medium TABLE 5. GI of a composite of four strains of control or heat-stressed, freeze-stressed, acid-stressed, and alkaline-stressed of Enterobacter sakazakii recovered on TSAP, LBDC, and differential, selective media using the ecometric technique Control Heat-stressed Freeze-stressed GI a Acid-stressed Alkaline-stressed All stress conditions TSAP 3.04 c 2.10 a 2.45 a 1.11 a 1.64 a 1.83 a LBDC 3.28 bc a a a a a 0.00 FCA 3.62 a b a b a b 0.19 DFI 3.05 c c c cd b d 0.96 OK 3.64 a bc bc bcd b c 0.79 VRBG 3.50 ab bc b cd b c 0.77 EE 3.03 c bc c d b d 0.94 RF 3.41 ab bc b bc b c 0.64 a Mean GI scores in the same column that are not followed by the same letter are significantly different (P 0.05). b Within control or stress condition, difference between the GI obtained on differential, selective media compared to the GI obtained on TSAP. dropwise manner, followed by thorough mixing. After storage for ca. 100 days, populations were reduced by ca. 1 log CFU/g and then subsequently by 2.5 (day 150), 3.0 (day 550), and ca. 3.3 (day 700) log CFU/g. Breeuwer et al. (4) desiccated E. sakazakii by transferring 50 l of exponential- or stationary-phase BHI cultures to 12-well culture plates, followed by drying at 25 C. Extrapolating from the subsequent survival curve for stationary-phase, the approximate numbers of desiccated inactivated by 30 and 46 days were 0 to 1.3 log CFU/ml and 1 to 1.5 CFU/ml, respectively, for the four test strains. Cells of one strain of exponential-phase of E. sakazakii were less resistant to desiccation stress. Caubilla- Barron et al. (6) studied the survival of nine strains of E. sakazakii following desiccation in freeze-dried and air-dried infant milk formula. Initial populations decreased by 2 log units immediately following desiccation and by an additional 4 log units during storage for 6 months at an unspecified temperature. Desiccation of used in our study and in the study reported by Edelson-Mammel and Buchanan (11) in powdered infant formula caused the death of fewer than reported in BHI cultures by Breeuwer et al. (4). Recovery of control by the ecometric technique. Analysis of a composite of all four strains of control revealed that E. sakazakii had a significantly higher GI on OK, FCA, VRBG, and RF than on DFI, TSAP, and EE (Table 5). Growth indices for DFI, TSAP, and EE were statistically equivalent to that of LBDC but significantly lower than indices for the four other media. The general order of performance of media in recovering control as determined by ecometric evaluation was OK, FCA, VRBG, RF LBDC, DFI, TSAP, EE. No significant differences in GI were noted in the performance of VRGB and RF, or in the performance of LBDC, DFI, TSAP, and EE. Although TSAP performed significantly better than selective media in recovering E. sakazakii using the spiral plating method, the medium was equivalent or inferior to LBDC and selective media in recovering control by the ecometric technique. We conducted nine replicate experiments to confirm these observations. Uyttendaele et al. (41) also found that TSA was inferior to selective media in terms of GI of some bacteria as determined using the ecometric technique. They reported that TSA was inferior to selective media in recovering Aeromonas sp. plated on Salmonella Shigella agar and Hektoen enteric agar and Klebsiella sp. plated on xylose lysine desoxycholate agar. The poor performance of TSAP in recovering control by the ecometric technique in our study may be due, in part, to the water-binding characteristics imparted by casein and soybean meal. Casein has a unique structure with distinct polar and hydrophobic domains (40). Casein and other ingredients may absorb water more quickly than would the ingredients in some of the other media, thereby removing the inoculum from the loop at a higher rate, which would result in a lower GI relative to GI values on less hydrophilic media. EE, which also contains casein, had a GI score similar to that of TSAP. This was unexpected in light of the exceptionally poor performance of EE and the superior performance of TSAP in the spiral plating study. Differences in water-absorbing capacities of the test media may not be evident using the spiral plating technique and may be a factor influencing the reliability of the ecometric technique to judge the performance of media in their abilities to support bacterial colony formation. The GI of TSAP for recovering control (3.04) was considerably lower following exposure of to freezing, heat, alkaline, and acid stress conditions (2.45, 2.10, 1.64, and 1.11, respectively) (Table 5). In experiments involving injured, TSAP performed better or was equivalent to selective media for recovering, which is contrary to the order of GI of media for recovering control but expected based on the performances of test media using the spiral plating technique. Differences in the performance of TSAP relative to other media in terms of GI scores for control and stressed are unexplainable. Recovery of heat-stressed by ecometric evaluation. Composite values for all four strains of heat-stressed were analyzed (Table 5). GI scores on TSAP and LBDC were significantly higher than the scores for all other media. The general order of performance in supporting recovery of heatstressed was TSAP, LBDC FCA, RF, OK, VRBG, EE, DFI. This order differs slightly from that obtained by spiral plating (TSAP LBDC, FCA OK, VRBG, DFI, EE) (Table 1). Recovery of freeze-stressed by the ecometric technique. Growth indices for all four strains of freeze-stressed were analyzed as a composite to determine the performance of each recovery medium (Table 5). Indices for TSAP, LBDC, and FCA were not significantly different but were significantly

7 VOL. 71, 2005 PERFORMANCE OF MEDIA FOR RECOVERY OF E. SAKAZAKII 7667 TABLE 6. GI of a composite of four strains of heat-stressed, freeze-stressed, acid-stressed, and alkaline-stressed of Enterobacter sakazakii recovered on TSAP and a composite of LBDC and differential, selective media determined using the ecometric technique Stress condition higher than indices obtained for other media. Growth indices of DFI and EE were significantly lower than those on all other media except OK. The general order of medium performance was TSAP, LBDC, FCA VRBG, RF, OK, DFI, EE. There were no significant differences, however, in the performance of OK, RF, and VRBG and in the performance of OK, DFI, and EE. The ecometric technique appeared to be more sensitive than spiral plating in discerning differences in performance of media for recovering freeze-stressed. Recovery of acid-stressed by the ecometric technique. Data for all four strains of acid-stressed were analyzed as a composite to determine the overall performance of each medium (Table 5). Growth indices for TSAP and LBDC were not significantly different but were significantly higher than for other media. The general order of performance was TSAP, LBDC FCA, RF, OK DFI, VRBG EE. There are similarities between this order and that obtained using the spiral plating technique, which gave a general order of performance of TSAP LBDC FCA, OK, VRBG, DFI, EE. Recovery of alkaline-stressed by the ecometric technique. Analysis of composite data for all four strains of alkaline-stressed revealed that the media were in the order TSAP, LBDC, FCA DFI, OK, VRBG, EE, RF for GI scores (Table 5). This order of performance is similar to that observed for spiral-plated alkaline-stressed, but spiral plating was somewhat more discerning, revealing that TSAP and LBDC were superior to FCA and that EE was inferior to all other media. Comparison of media for recovering exposed to a composite of all stress conditions by the ecometric technique. Data from ecometric evaluations using heat-, freeze-, acid-, and alkaline-stressed were combined and analyzed for significant differences in GI scores for each test medium (Table 5). The order of performance was TSAP, LBDC FCA RF, VRBG, OK DFI, EE. While this order is similar to that observed for spiral plating, the performances of specific media used in the two methods were not always the same. Data from ecometric evaluation were analyzed by combining GI scores for the four strains of E. sakazakii as a composite (Table 6). The order of GI for control and stressed on TSAP was control freezing thermal alkaline acid. Data from spiral plating, on the other hand, showed that the general order of recovery on TSAP as affected by stress condition was control alkaline thermal acid freezing, although there were no significant differences among the number of thermal-, acid-, and alkaline-stressed recovered (Table 2). When plated on LBDC and differential, selective media using the ecometric technique, the order of GI according to stress conditions was control freezing thermal, alkaline acid (Table 6). This differs from the order obtained from spiral plating (control alkaline thermal acid, freezing) (Table 2). The ecometric technique has been reported by some researchers to be a consistent and efficient means of testing the performance of media (22, 30, 41). When drawing conclusions concerning the performance of media in recovering using the ecometric technique, consideration should be given not only to the ability of the medium to support colony development, but also to the chemical and physical nature of the surface of the medium. Chemical characteristics, such as hydrophobicity, may affect the rate at which are removed from the inoculating loop, thus influencing the GI score. Our study indicates that, overall, direct (spiral) plating is superior to the ecometric technique to determine the relative performances of media for recovering healthy and injured of E. sakazakii. Appearance of colonies on test media. Colonies formed by the four strains of E. sakazakii on TSAP, LBDC, and differential, selective media incubated at 37 C for 24 h were examined for color, size, and overall appearance. Strain ES132 formed consistently larger colonies than did the other strains on all media. Strain was the only strain that produced matte colonies rather than mucoid colonies on TSAP, VRBG, and EE. All strains produced yellow pigmentation, to varying degrees, on TSAP. Only strain 4921 lightened the color of FCA. Growth of strain 4921 on EE was very weak, forming punctiform colonies. Only strain produced colonies with a rubbery consistency on TSAP, OK, LBDC, and EE. Colonies of strains and 4921 had rubbery consistencies on VRBG, DFI, FCA, and RF. Because data from direct plating of samples are generally considered a standard against which observations from ecometric evaluation of media are measured, spiral plating, in this study, should be considered more definitive and counts should receive greater weight than GI scores when judging the performance of media for supporting the growth of healthy and injured E. sazkazakii. Other studies, although recognizing the efficiency and utility of the ecometric technique as a tool to evaluate media, have also concluded that standard dilution, direct-plating, and colony-counting techniques have a higher level of accuracy than the ecometric technique (8, 30, 44). Even without evaluating the performance of RF medium using the spiral-plating technique, some conclusions can still be drawn concerning its performance in comparison to the seven other media based on observations from ecometric evaluation. Because ecometric evaluation showed that RF VRBG, OK DFI, EE, it is concluded that RF medium performs equivalently or better than VRBG, OK, DFI, and EE in recovering injured E. sakazakii. Colonies of E. sakazakii formed on GI a TSAP Differential, selective media c R d Control b 3.04 a a 3.39 a 0.35 Thermal a 2.09 c 0.95 b 1.34 c Freeze a 2.45 b 0.59 b 1.69 b Acid a 1.11 e 1.93 b 0.51 d Alkaline a 1.64 d 1.40 b 1.30 c a Mean GI scores in the same column that are not followed by the same letter are significantly different (P 0.05). Mean GI scores in the same row that are not preceded by the same letter are significantly different. b Within TSAP or within composites of differential, selective media, difference in GI obtained for stressed and the GI obtained for control. c Includes LBDC. d Difference between the GI obtained on differential, selective media and the GI obtained on TSAP.

8 7668 GURTLER AND BEUCHAT APPL. ENVIRON. MICROBIOL. orange-colored RF agar have a distinct black/dark-blue color. Based on the ease of detecting colonies on the basis of chromogenic/fluorogenic characteristics, RF agar, along with LBDC and DFI, shows promise. Iversen et al. (18) tested 17 genera comprising 148 non-e. sakazakii strains, excluding Shigella, in the family Enterobacteriaceae for growth and colony appearance on DFI medium. They observed that 12.8% of the strains were false positive for E. sakazakii on DFI. In a companion study (unpublished), we observed that one of eight strains of Shigella sonnei was false positive for E. sakazakii on DFI. In another study, Iversen and Forsythe (20) analyzed 82 powdered infant formula products and 404 other food products for the presence of E. sakazakii using DFI medium. False-positive presumptive colonies of E. sakazakii were observed at a level of 38.5% on DFI medium compared to 72.9% on TSA after enrichment in buffered peptone water and EE broth and plating on VRBG agar. The use of chromogenic agars, such as RF and DFI, or fluorogenic media, such as OK and LBDC, decreases the time to detection of presumptive-positive E. sakazakii by 24 h over the traditional media recommended in the FDA method (42). However, the relative performances of differential, selective media in recovering stressed E. sakazakii must be considered in any method recommended for analysis of powdered infant formula and other foods. The poor performance of EE agar in our study clearly raises a concern about the ability of EE broth, which is used in the FDA method (42), to recover not only injured but also healthy of E. sakazakii. 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