Simultaneous Enrichment of Shiga Toxin-Producing Escherichia coli O157 and O26 and Salmonella in Food Samples Using Universal Preenrichment Broth

Abstract

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin-producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42 degrees C recovered significantly more cells from inoculated beef than UPB at 35 degrees C and from radish sprout samples than UPB at 35 degrees C and mEC+n. With regard to Salmonella, UPB incubated at 42 degrees C was as effective as UPB at 35 degrees C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42 degrees C and UPB at 35 degrees C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42 degrees C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42 degrees C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42 degrees C.

Full text

Simultaneous Enrichment of Shiga Toxin–Producing Escherichia
coli O157 and O26 and Salmonella in Food Samples Using
Universal Preenrichment Broth
MASASHI KANKI,*KAZUKO SETO, JUNKO SAKATA, TETSUYA HARADA, AND YUKO KUMEDA
Division of Bacteriology, Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan
MS 09-153: Received 7 April 2009/Accepted 30 May 2009
ABSTRACT
Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mECzn) for
enrichment of Shiga toxin–producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of
Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal,
freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42uC recovered significantly more cells from
inoculated beef than UPB at 35uC and from radish sprout samples than UPB at 35uC and mECzn. With regard to Salmonella,
UPB incubated at 42uC was as effective as UPB at 35uC and BPW at recovering cells from beef and radish sprout samples. No
significant difference was noted between the effectiveness of UPB at 42uC and UPB at 35uC or BPW in the recovery of
Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42uC, one O157:H7 strain was isolated from the
mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen
inoculation. The present study found that UPB incubated at 42uC was as effective as, or better than, mECzn for enrichment of
O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time,
samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42uC.
Shiga toxin–producing Escherichia coli (STEC) organ-
isms have been associated with a number of life-threatening
diseases, such as hemorrhagic colitis and hemolytic uremic
syndrome (4). The major hosts for STEC are ruminants, and
STEC is known to be transmitted by several routes,
including food and water (6). A 2007 study showed that
STEC of serogroup O157 and O26 accounted for 87.6%
(75.0 and 12.5%, respectively) of STEC isolated from
humans in Japan (14).Salmonella enterica is a major cause
of foodborne infection prevalent in many types of food,
particularly eggs, poultry, and meat (13). Because E. coli
O157 and Salmonella are considered to be the foodborne
pathogens meriting the most attention at present, and
because they may be found in a wide range of food
products, many food samples undergo screening for the
presence of these two bacterial species (1, 19, 20, 25).
However, the first-stage enrichment broth and cultivation
temperature ideal for STEC differ from the conditions ideal
for Salmonella, resulting in increased labor and monetary
costs associated with having to test samples separately.
Modified E. coli broth (mEC) and Trypticase soy broth
(TSB) with or without supplements (cefixime, tellurite, and
novobiocin) are frequently used as enrichment broth for
STEC (27), whereas buffered peptone water (BPW) and
lactose broth are mainly used as preenrichment broth in
standard culture methods for detecting Salmonella, such as
the Bacteriological Analytical Manual (26) and the
International Organization for Standardization (15) meth-
ods. Standard cultivation temperatures are 37 to 42uC for
STEC and 35 to 37uC for Salmonella. In Japan, O157 and
O26 E. coli strains are cultivated in mEC supplemented with
novobiocin (mECzn) at 42uC(10) as the standard
enrichment broth, while Salmonella is cultivated in BPW
at 35uC(2, 23).
To enable the simultaneous enrichment of Salmonella
and Listeria, universal preenrichment broth (UPB), which is
highly buffered and low in carbohydrates, was developed.
This medium hampers pH reduction caused by heavy
microbial growth, enabling repair of injured cells (3). Given
the medium’s resuscitative properties and the fact that O157
may be sublethally injured or stressed by conditions in the
food environment or during food processing and storage (7),
several reports have cited the usefulness of UPB in detecting
STEC O157 in food samples (16, 24, 28). However, these
studies observed O157 cultivated in UPB at an original
direction temperature of 35uC, whereas recent reports have
determined that O157 has an optimal growth temperature of
40uC(27).
Here, we compared the effectiveness of UPB incubated
at 35 and 42uC with mECzn for enrichment of O157 and
O26, and with BPW for preenrichment of Salmonella in
artificially contaminated beef and radish sprout samples. We
also assessed the effectiveness of UPB incubated at 35 and
42uC versus mECzn for detection of O157 and O26 in
naturally contaminated beef and pork samples and versus
BPW for detection of Salmonella in naturally contaminated
poultry samples. Simultaneous enrichment of STEC O157
* Author for correspondence. Tel: 81-6-6972-1321; Fax: 81-6-6972-1329;
E-mail: kanki@iph.pref.osaka.jp.
2065
Journal of Food Protection, Vol. 72, No. 10, 2009, Pages 2065–2070
Copyright G, International Association for Food Protection
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and O26 and Salmonella using UPB incubated at 42uC
would reduce the amount of time, labor, and incubator space
required for food analyses.
MATERIALS AND METHODS
Bacteria. Ten strains each of STEC O157 (seven strains of
O157:H7 and three strains of O157:HNM [nonmotile]), STEC O26
(eight strains of O26:H11 and two strains of O26:HNM), and S.
enterica (five strains each of serovars Enteritidis and Typhimur-
ium) were used to artificially inoculate food samples. The strains
were isolated from patients in Osaka, Japan, from 1996 to 2008.
Food samples and sample preparation. From February
2008 to March 2009, beef, radish sprout, cattle offal, pork, and
poultry samples were obtained from local retail stores in Osaka,
Japan. Before inoculation, beef and radish sprout samples were
first confirmed to be STEC free by loop-mediated isothermal
amplification (LAMP) assay (22) and Salmonella free by using the
culture procedure described below. A total of 59 beef samples
including 6 offal samples and 50 pork samples were examined for
natural contamination with O157 and O26. A total of 205 poultry
samples, including 26 giblet samples, were examined for natural
contamination with Salmonella. All samples, both animal and
vegetable, were cut into pieces approximately 2 cm in length. The
samples were then thoroughly mixed and separated into groups of
three 25-g portions each.
Thermal treatment for STEC O157 and O26 and
Salmonella.The bacterial strains were first injured by thermal
treatment as referenced in the study by Jiang et al. (16). Briefly,
samples were cultivated for 18 h in 2 ml of TSB (Difco, Becton
Dickinson, Sparks, MD) and then diluted to 10
8
CFU/ml with
phosphate-buffered saline (PBS). A 1-ml dilution was added to
99 ml of TSB preheated to 55uC in a 200-ml Erlenmeyer flask, and
the flask was held at 55uC in a water bath until 10
3
CFU of heat-
injured cells per ml were obtained from 10
6
CFU of the test strains
per ml (Tables 1 and 2). For O157 and O26 inoculation in radish
sprouts, however, a dilution of 10
4
CFU of heat-injured cells per
ml was prepared. After this heat treatment, the flask was cooled on
ice for 10 min. The bacterial suspensions were then diluted with
PBS and added to three 25-g portions of either beef or radish
sprout samples in stomacher bags at the appropriate level for
inoculation (Tables 1 and 2). The number of heat-injured cells was
calculated by serial dilution and surface spread plating on
Trypticase soy agar (TSA).
Freezing treatment and no treatment for STEC O157 and
O26 and Salmonella.Samples were cultivated in 2 ml of TSB for
18 h and then diluted into 10 CFU/ml with PBS. A 100-ml dilution
was added to three 25-g portions each of beef and radish sprout
samples in stomacher bags. For the freezing treatment, the
inoculated beef samples were stored in a freezer at 220uC for 4
weeks.
Culture procedure for STEC O157 and O26. Two 25-g
portions each of artificially contaminated beef and radish sprout
samples and naturally contaminated beef and pork samples were
added to 225 ml of UPB (Difco, Becton Dickinson), and one 25-g
portion each was added to mECzn (Eiken Chemical Ltd., Tokyo,
Japan). The samples were homogenized for 1 min with a Pro-
media SH-001 stomacher (Elmex Ltd., Tokyo, Japan). One of the
UPB homogenates and the mECzn homogenate were incubated at
42uC for 20 to 24 h, while the other UPB homogenate was
incubated at 35uC for 20 to 24 h. Following incubation of the
artificially contaminated samples, STEC O157 and O26 were
detected by LAMP assay, plating, and immunomagnetic separation
(IMS). Enrichment cultures of naturally contaminated samples
were screened for STEC by LAMP assay. LAMP-positive samples
were processed by IMS assay for O157 and O26 and then spread
onto TSA at a 10
6
-fold dilution for colony hybridization.
LAMP assay for O157 and O26. To assess the presence of
Shiga toxin genes (stx) in the enriched samples, the LAMP assay
was performed using the Loopamp Verotoxin-producing Esche-
richia coli Detection Kit (Eiken Chemical). Fifty microliters of
cultured medium was mixed with 50 ml of a solution for DNA
extraction and heated at 95uC for 5 min. After centrifugation at
16,000 |gfor 1 min, 5 ml of supernatant was mixed with 20 mlof
reaction mixture in the reaction tube and reacted at 65uC for 1 h
using the Loopamp Realtime Turbidimeter LA-320C (Eiken
Chemical). Using this equipment, the turbidity that the LAMP
reaction caused proportional to the amount of DNA amplified was
detected in real time.
Plating method for O157 and O26. A loopful of the
enriched samples was streaked onto sorbitol MacConkey agar
(Oxoid Ltd., Hampshire, UK) to test for O157 and rhamnose
MacConkey agar to test for O26, both supplemented with 0.05 mg
of cefixime per liter and 2.5 mg of potassium tellurite per liter
(Aska Diagnostics Inc., Tokyo, Japan) (CT-SMAC and CT-
RMAC, respectively). After incubation at 36uC for 18 to 24 h,
presumed colonies were confirmed by the agglutination test using
E. coli O157 or O26 antiserum (Denka Seiken Ltd., Tokyo, Japan).
IMS method for O157 and O26. One milliliter of the
enriched samples was mixed with one drop (approximately 25 ml)
of immunomagnetic beads coated with O157 or O26 antibody
(Denka Seiken Ltd.). After the mixture was allowed to react at
room temperature for 30 min, the beads were separated using a
magnetic particle concentrator (Dynal MPC-S, Invitrogen, Carls-
bad, CA), washed with 1 ml of PBS, separated again, and then
suspended in 0.1 ml of PBS. Thirty microliters of bead suspension
was streaked onto a CT-SMAC and CT-RMAC plate and
incubated at 36uC for 18 to 24 h. Presumed colonies were
confirmed by the agglutination test using E. coli O157 or O26
antiserum (Denka Seiken Ltd.).
Colony hybridization assay. Colonies on TSA were lifted to
Hybond-Nzmembranes (GE Healthcare UK Ltd., Buckingham-
shire, UK). The membranes were prehybridized in ExpressHyb
hybridization solution (Clontech, Takara Bio Inc., Shiga, Japan) at
50uC for 30 min and then hybridized at 50uC for 2 h in the same
solution containing an stx DNA probe. The probe was generated
using the primers of Karch and Meyer (18) and the PCR DIG
Probe Synthesis Kit (Roche Ltd., Basel, Switzerland). The
hybridized probe on the membrane was detected by alkaline
phosphatase–conjugated anti-digoxigenin antibody (Fab; Roche).
The enzyme-catalyzed color reaction was carried out with a
nitroblue tetrazolium salt–5-bromo-4-chloro-3-indolylphosphate
(Roche) system.
Culture procedure for Salmonella.Two 25-g portions each
of artificially contaminated beef and radish sprout samples and
naturally contaminated poultry samples were added to 225 ml of
UPB, and one 25-g portion each was added to 225 ml of BPW
(Eiken Chemical Ltd.). The samples were homogenized with a
stomacher for 1 min. For all samples, one of the UPB homogenates
and the BPW homogenate were incubated at 35uC for 20 to 24 h,
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TABLE 1. Comparison of Shiga toxin–producing Escherichia coli O157 and O26 detection by loop-mediated isothermal amplification (LAMP), plating, and immunomagnetic separation (IMS) in
beef and radish sprout samples after cultivation under three enrichment conditions
Treatment
conditions
Inoculum dose
(CFU/25 g)
UPB at 42uC UPB at 35uCmECzn
LAMP Plating IMS Total LAMP Plating IMS Total LAMP Plating IMS Total
Beef
No treatment
O157 None 1.4–2.8 9/10
a
8/10 9/10 26/30 8/10 5/10 8/10 21/30 10/10 9/10 10/10 29/30
O26 None 1.1–2.4 8/10 9/10 10/10 27/30 9/10 9/10 9/10 27/30 9/10 9/10 9/10 27/30
Thermal treatment
O157 55uC, 35 min 2.7–9.8 7/10 9/10 10/10 26/30 A
b
0/10 2/10 2/10 4/30 AB 9/10 6/10 8/10 23/30 B
O26 55uC, 20 min 1.5–9.3 4/10 3/10 8/10 15/30 A0/10 0/10 2/10 2/30 AB 9/10 4/10 7/10 20/30 B
Freezing treatment
O157 220uC, 4 wk 1.4–2.7 5/10 8/10 8/10 21/30 AB 3/10 0/10 4/10 7/30 A4/10 3/10 4/10 11/30 B
O26 220uC, 4 wk 1.0–2.0 8/10 9/10 10/10 27/30 AB 2/10 4/10 4/10 10/30 A5/10 5/10 5/10 15/30 B
Radish sprouts
No treatment
O157 None 1.0–1.9 10/10 5/10 9/10 24/30 AB 6/10 3/10 5/10 14/30 A6/10 2/10 4/10 12/30 B
O26 None 1.0–1.8 7/10 6/10 7/10 20/30 A5/10 1/10 4/10 10/30 A5/10 5/10 6/10 16/30
Thermal treatment
O157 55uC, 15 min 120–810 9/10 5/10 9/10 23/30 A7/10 3/10 7/10 17/30 B2/10 1/10 1/10 4/30 AB
O26 55uC, 15 min 200–860 10/10 9/10 10/10 29/30 AB 7/10 1/10 5/10 13/30 A4/10 4/10 6/10 14/30 B
a
Number of positive samples/number of samples tested.
b
Total numbers followed by the same letter are significantly different (P,0.01).
J. Food Prot., Vol. 72, No. 10 OPTIMAL CONDITIONS FOR SIMULTANEOUS ENRICHMENT OF STEC AND SALMONELLA 2067
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while the other UPB homogenate was incubated at 42uC for 20 to
24 h. After this incubation, 0.1 ml each of the cultivated broths
was added to 10 ml of Rappaport-Vassiliadis broth (Oxoid Ltd.)
and incubated at 42uC for 20 to 24 h. A loopful of the cultures was
then streaked onto xylose lysine deoxycholate agar (Difco) and
brilliant green agar (Oxoid Ltd.) supplemented with sulfapyridine
(Sigma-Aldrich Co., St. Louis, MO), followed by incubation at
35uC for 20 to 24 h. Presumed Salmonella colonies were
confirmed by the agglutination test using Salmonella O and H
antisera (Denka Seiken Ltd.).
Statistical analysis. To evaluate the success of cultivation
under the three enrichment conditions for each pathogen and
treatment type, data regarding the cells detected in artificially
inoculated beef and radish sprout samples were analyzed using
Fisher’s exact test. With regard to detection of O157 or O26 cells,
the total detection numbers obtained by LAMP, plating, and IMS
were compared for every 3 of 10 samples. Data regarding
Salmonella detected in naturally contaminated chicken samples
were analyzed using the x
2
test. The level of significance for both
tests was set at Pvalues of ,0.01. Data regarding O157 or O26
detected in naturally contaminated beef and pork samples were not
statistically analyzed because only one positive sample was
obtained.
RESULTS
Recovery of inoculated STEC O157 and O26 strains
from beef samples. With regard to detection of uninjured
O157 and O26 cells, 10 inoculated beef samples were
initially divided into three portions each, and each portion
per sample was enriched in either UPB at 42uC, UPB at
35uC, or mECzn. Positive results were obtained by the
three detection methods in 26 and 27 samples enriched in
UPB at 42uC, 21 and 27 samples enriched in UPB at 35uC,
and 29 and 27 samples enriched in mECzn, respectively
(Table 1). The number of uninjured O157 and O26 cells
detected in beef samples did not significantly differ between
the three enrichment conditions (P.0.01).
With regard to detection of heat-injured O157 and O26
cells, positive results were obtained in 26 and 15 samples
enriched in UPB at 42uC, 4 and 2 samples enriched in UPB
at 35uC, and 23 and 20 samples enriched in mECzn,
respectively. Although a significant difference in detection
of heat-injured O157 and O26 cells was observed between
UPB at 35uC and the other enrichment conditions, no
difference was observed between enrichment in UPB at
42uC and enrichment in mECzn.
With regard to detection of freeze-injured O157 and
O26, positive results were obtained in 21 and 27 samples
enriched in UPB at 42uC, 7 and 10 samples enriched in UPB
at 35uC, and 11 and 15 samples enriched in mECzn,
respectively. A significant difference in detection of freeze-
injured O157 and O26 cells was observed between UPB at
42uC and the other enrichment conditions.
Recovery of inoculated STEC O157 and O26 strains
from radish sprout samples. With regard to detection of
uninjured O157 and O26 cells, 10 inoculated radish sprout
samples were initially divided into three portions each, and
each portion per sample was enriched in either UPB at
42uC, UPB at 35uC, or mECzn. Positive results were
obtained by the three detection methods in 24 and 20
samples enriched in UPB at 42uC, 14 and 10 samples
enriched in UPB at 35uC, and 12 and 16 samples enriched
in mECzn, respectively (Table 1). A significant difference
was observed between UPB at 42uCandtheother
enrichment conditions with regard to detection of unin-
juredO157inradishsproutsandbetweenUPBat42uCand
UPB at 35uCwithregardtodetectionofuninjuredO26(P
,0.01).
With regard to heat-injured O157 and O26 cells,
positive results were obtained for 23 and 29 samples
enriched in UPB at 42uC, 17 and 13 samples enriched in
UPB at 35uC, and 4 and 14 samples enriched in mECzn,
respectively. A significant difference was observed between
mECzn and the other enrichment conditions with regard to
detection of heat-injured O157 and between UPB at 42uC
and the other enrichment conditions with regard to detection
of heat-injured O26 cells.
Recovery of inoculated Salmonella strains from beef
and radish sprout samples. Of 10 beef samples inoculated
with Salmonella, heat-injured cells were isolated from 10, 9,
and 10 samples preenriched in UPB at 42uC, UPB at 35uC,
and BPW, respectively, and freeze-injured cells were
isolated from 7, 9, and 8 samples preenriched under the
same conditions (Table 2). No significant difference was
observed between the three enrichment conditions (P.
0.01).
TABLE 2. Comparison of Salmonella detection in beef and radish sprout samples under three enrichment conditions
Inoculum dose Treatment conditions UPB at 42uC UPB at 35uC BPW
Beef
Thermal treatment
1.2–9.5 55uC, 15 min 10/10
a
9/10 10/10
Freezing treatment
1.6–5.2 220uC, 4 wk 7/10 9/10 8/10
Radish sprouts
No treatment
1.1–6.1 None 9/10 7/10 9/10
Thermal treatment
11–61 55uC, 10 min 6/10 8/10 3/10
a
Number of positive samples/number of samples tested.
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Of 10 radish sprout samples preenriched under the same
conditions as above, heat-injured Salmonella was isolated
from 6 and 8 samples preenriched in UPB at 42 and 35uC,
respectively, and from 3 samples enriched in BPW. No
significant difference was observed between the three
enrichment conditions. In total, uninjured Salmonella cells
were isolated from seven to nine samples for each broth.
Isolation of STEC O157 and O26 from naturally
contaminated beef and pork samples. Of the six cattle
offal samples, two (33%) (cattle liver and mixed offal)
under the three enrichment conditions were found to be
positive for stx genes by LAMP assay. One strain was
recovered from the mixed offal sample by IMS assay for
O157 and belonged to serotype O157:H7. Another strain
was recovered by colony hybridization and belonged to
serotype O8:H19. Of 53 beef samples, 1 (1.9%) enriched in
UPB at 42uC was found to be positive for stx genes. One
strain was recovered by colony hybridization and belonged
to serotype O113:H34. No positive results were obtained by
LAMP assay from any of the 50 pork samples.
Isolation of Salmonella from naturally contaminat-
ed poultry samples. Of the 205 poultry samples analyzed,
Salmonella was isolated from 112 samples enriched in UPB
at 42uC (54.6%), from 101 samples enriched in UPB at
35uC (49.3%), and from 104 samples enriched in BPW
(50.7%). No significant difference was observed between
the three enrichment conditions (P.0.01).
DISCUSSION
In this study, we evaluated the effectiveness of UPB
incubated at 42uC in detecting STEC O157 and O26 and
Salmonella in beef, radish sprout, and poultry samples and
compared it with conventional detection methods. Enrich-
ment in UPB at 42uC was more effective in detecting O157
and O26 in beef and radish sprout samples than enrichment
in mECzn, and it was comparable to enrichment in BPW in
detecting Salmonella in food samples. Overall, our findings
indicate that UPB at 42uC is capable of simul-
taneous enrichment of O157 and O26 and Salmonella.
In their study, Fratamico and Bagi (7) demonstrated
that the odds of obtaining a positive result for O157 from
ground beef increased 10-fold when samples were enriched
in mECzn at 42 versus 35uC. Further, Hara-Kudo et al. (11)
reported that enrichment in mECznat42uC was more
effective at isolating O157 and O26 from both ground beef
and radish sprouts than enrichment at 37uC. Similarly,
enrichment in UPB at 42uC is considered more effective at
recovering O157 and O26 than enrichment at 35uC.
With regard to freeze-injured O157 and O26 cells in
beef and heat-injured cells in radish sprouts, enrichment in
UPB at 42uC proved significantly more effective at
detecting cells than enrichment in mECzn. Given that
UPB was initially developed for the detection of injured
Salmonella and Listeria (3), UPB was predicted to be
superior to mECzn in detecting injured cells. The present
study further noted, however, that UPB at 42uC was also
effective in the detection of uninjured O157 and O26 cells in
radish sprout samples. The lower performance observed in
detecting uninjured O157 and O26 in radish sprouts
compared with that in beef samples suggests that enrich-
ment conditions were harsher in the radish sprout samples,
hampering resuscitation. Hammack et al. (8) reported that
orange juice samples enriched with UPB yielded better
recovery of inoculated Salmonella than samples enriched
with lactose broth. UPB is also believed to be effective at
detecting cells in foods that influence resuscitation and
multiplication of O157 and O26. Under mild conditions, as
in the enriched beef samples, we observed no significant
difference in the recovery of heat-injured and uninjured cells
between UPB at 42uC and mECzn. STEC O157 was
isolated from naturally contaminated cattle offal enriched in
UPB at 42uC, albeit only one strain. Our finding that
detection using UPB at 42uC was comparable to, or better
than, that of mECzn suggests that enrichment in UPB at
42uC may be effective at detecting O157 and O26 in food
samples.
Preenrichment before inoculation of enrichment media
for Salmonella was first reported in 1961 (21), and BPW
was reported as a preenrichment broth for sublethally
injured Salmonella in frozen meat samples in 1973 (5).
Since these reports, the incubation temperature of preen-
richment broth for Salmonella has been universally accepted
to be between 35 and 37uC. However, Heinonen-Tanski et
al. (12) demonstrated that an incubation temperature of
43uC was better than 37uC for BPW with ferrioxamine E.
Given our finding that the effectiveness of UPB at 42uC was
equal to that of both UPB at 35uC and BPW for the recovery
of Salmonella, we speculate that incubation temperature is
not a critical determinant for preenrichment of Salmonella.
UPB at 42uC showed high sensitivity on naturally
contaminated poultry samples, in which Salmonella counts
are frequently less than 1 CFU/g (2, 17), an observation
which suggests that UPB at 42uC is effective for preen-
richment of Salmonella in food samples.
Enrichment methods for the detection of STEC have
been modified in various ways. In particular, antibiotics are
often added to enrichment broth to facilitate the growth of
STEC by limiting the growth of background microflora
(27). However, novobiocin and bile salts have been reported
to inhibit resuscitation of injured O157 cells (9). UPB is
believed to be mild enough to allow for resuscitation of
antibiotic-sensitive cells owing to its containing no selective
reagents, allowing for preparation of a simple medium
without the need for supplements. Detection analyses for
O157, O26, and Salmonella have been conducted with a
large range of food products for as long as these organisms
have been a public health priority. Based on the findings of
the present study, we believe that UPB incubated at 42uCis
sufficient for the simultaneous enrichment of O157 and O26
and Salmonella obtained from food samples, an enrichment
method which will reduce the labor, time, volume of
samples, and space required to conduct these analyses.
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2070 KANKI ET AL. J. Food Prot., Vol. 72, No. 10
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