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Journal of Clinical Microbiology, December 1999, p. 4120-4123, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Recovery of Salmonella by Using Selenite
Brilliant Green Sulfa Enrichment Broth
Chiao-tang
Chang,1,2
Chung-Yee
Yuo,3
Hui-Ching
Shen,4
A-Mai
Li,1
Chao-yu
Chen,1
Jui-ling
Chou,1 and
Shiao-ping
Huang4,*
Clinical Laboratory1
and Department of Medical Research,2
Yuan's General Hospital, Kaohsiung 802, Department of
Biology, Kaohsiung Medical University, Kaohsiung
807,3 and Department of Medical
Technology, Fooyin Institute of Technology, Ta-Liao Hsiang,
Kaohsiung Hsien 831,4 Taiwan, Republic of China
Received 7 May 1999/Returned for modification 3 July 1999/Accepted 23 August 1999
 |
ABSTRACT |
The efficacy and sensitivity of selenite brilliant green sulfa
enrichment (SBG) broth for the isolation of Salmonella from fecal specimens were evaluated by using both clinical and artificially infected (artificial) fecal specimens. An examination of 1,588 clinical
fecal specimens found Salmonella in 296 specimens,
including 89 cases detected by the direct-plating
xylose-lysine-desoxycholate method and an additional 207 cases detected
after enrichment with SBG broth. Therefore, the recovery of
Salmonella with SBG broth is increased 3.3-fold over that
by the direct-plating method alone. Furthermore, the isolation rate of
Salmonella is higher when using SBG broth than when using
gram-negative (GN) broth or GN broth supplemented with sodium selenite.
To determine the sensitivity for the recovery of
Salmonella, artificial specimens containing various amounts
of Salmonella were prepared and analyzed. The results
indicated that the sensitivity is also higher with SBG broth than with
GN broth. Moreover, the optimal incubation period for SBG broth can be
extended to 24 h. In conclusion, the SBG enrichment method
provides a higher recovery rate of Salmonella from fecal specimens.
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TEXT |
Enrichment is a critical step in
enhancing the growth of certain bacterial species while inhibiting the
development of unwanted microorganisms. Enrichment broths are most
commonly used in clinical laboratories for the recovery of pathogens
from fecal specimens. The direct-plating and enrichment methods have
been combined for the best isolation of pathogens from patients with
diarrhea (9). Several kinds of enrichment broth are
particularly helpful in the recovery of pathogens from the stools of
Salmonella carriers or patients with slight
Shigella infections in whom the number of pathogens may be
as low as 100/g of feces, whereas Escherichia coli or other
enteric bacilli may reach massive concentrations, as high as
109/g of feces (4-8, 12, 13).
The recovery of Salmonella from various specimens with a
variety of media and incubation conditions has been documented (1, 3, 5-7, 9-14). Recently, the efficacies of several new
selective media for the isolation of Salmonella have been
compared. These media include Hektoen enteric agar (HE), Rambach
agar, SM-ID medium, xylose-lysine-tergitol 4 agar (XLT4),
novobiocin-brilliant green-glycerol-lactose agar, and modified
semisolid Rappaport-Vassiliadis medium. In particular, the results
indicated that the sensitivity for the isolation of
Salmonella was dramatically increased by the utilization of
tetrathionate enrichment broth (2). Moreover, the
effectiveness of three enrichment broths, selenite cystine broth,
tetrathionate broth, and Rappaport-Vassiliadis medium, for the recovery
of Salmonella from contaminated food specimens has also been
evaluated (7).
Xylose-lysine-desoxycholate (XLD) agar is the most commonly used highly
selective medium for the recovery of enteric pathogens from fecal
specimens (15). In addition, enrichment broths are usually
used to enhance the recovery of Salmonella species from fecal specimens. The two most frequently used enrichment broths are
gram-negative (GN) broth and selenite F (SF) broth. If these broths are
used, the subculture should be performed within 4 to 6 h for GN
broth and within 8 to 12 h for SF broth. Selenite brilliant green
sulfa (SBG) broth, originally designed for the detection of
Salmonella in egg and food specimens, was previously used by us to perform enrichment cultures of fecal specimens. In particular, the culture period for enrichment can be extended to 24 h
(12). This study was undertaken to evaluate the efficacy and
sensitivity of the SBG enrichment broth for the isolation of
Salmonella from both clinical and artificially infected
(artificial) fecal specimens.
Clinical specimens.
Clinical specimens were collected from
patients suspected of having gastrointestinal tract infection in
Yuan's General Hospital, Kaohsiung, Taiwan. Between January 1995 and
December 1997, 1,588 specimens were obtained from the pediatric
department. In addition, another 155 specimens were collected from the
outpatient department from April to July 1997.
Bacteria and artificial specimens.
To determine the
sensitivity of enrichment broths, various amounts of group B and group
D Salmonella spp. were added to stool to create the
artificial specimens. All bacteria were obtained from clinical isolates
and identified by biochemical and serological tests. The amount of
bacteria added was adjusted depending on the bacterial turbidity by
using the McFarland standard.
Enrichment broths and selective media.
Four enrichment broths,
GN broth, GN broth supplemented with sodium selenite (SGN broth), SF
broth, and SBG broth, were used. The isolation of Salmonella
spp. was performed by streaking the stool specimen on plates containing
the selective medium, XLD agar. The GN broth, SF broth, and XLD agar
were purchased from Difco Laboratories (Detroit, Mich.). Sodium
selenite (E. Merck, Darmstadt, Germany) was added to the SBG broth
(Eiken Chemical Co., Tokyo, Japan).
Direct plating and subculture with XLD agar.
To isolate
Salmonella spp., all clinical specimens were inoculated onto
XLD plates and into enrichment broth and then incubated overnight at
35°C in a CO2 incubator. The subculture was performed from enrichment broth to another XLD agar plate within the optimal incubation periods of each broth, 4 to 6 h for GN broth and 8 to
12 h for SF broth (for a review, see reference
8).
Identification of bacteria.
The identification media, triple
sugar iron agar, Simmons citrate agar, Christensen's urea agar,
sulfide-indole motility medium, ornithine decarboxylase, and oxidase
reagent were purchased from Difco Laboratories. The semisolid VP medium
was from Eiken Chemical Co. (Tokyo, Japan). The API 20E identification
system (bioMerieux Vitek, Inc., Hazelwood, Mo.) and serological
identification kit (Difco Laboratories) were used as the reference
methods to identify Salmonella species. All media were
prepared and used in accordance with the manufacturer's descriptions.
As shown in Table 1,
Salmonella spp. were isolated in 296 clinical cultures from
1,588 specimens (18.6%). Only 89 positive cases (5.6%) were isolated
by using the direct-plating method. The additional 207 positive cases
(13%) were isolated by using SBG broth as the enrichment broth. This
striking result indicates that the combination of direct plating with
SBG enrichment increased the isolation rate 3.3-fold. Therefore, the
use of SBG broth will help increase the isolation of
Salmonella spp. from fecal specimens.
To compare the isolation rates of Salmonella spp. between
SBG and GN enrichment broths, another 155 clinical fecal specimens were
analyzed. Since the optimal enrichment periods are 4 to 6 h for GN
broth and 16 to 24 h for SBG broth, we used the incubation periods
of 6 h for GN broth and 16 h for SBG broth. The results shown
in Table 2 indicate that the isolation
rates of Salmonella spp. are 10.3% for SBG broth and 3.8%
for GN broth. This result reveals that the isolation rate of
Salmonella spp. is higher with SBG enrichment broth than
with GN broth. To clarify the effect of sodium selenite on the
isolation rate of Salmonella spp., sodium selenite was added
to GN broth as the supplement. Ninety-four clinical specimens were used
to examine the isolation rate of Salmonella spp. The results
in Table 3 show that the isolation rate
is higher with GN broth supplemented with sodium selenite than with GN
broth alone. However, both GN broth and GN broth supplemented with
sodium selenite have lower isolation rates than that of SBG broth.
To examine the optimal culture periods for various enrichment broths,
artificial specimens were used. Two different culture
periods, 6 and
24 h, were chosen for use in subculture. The results
shown in Fig.
1 indicate that GN broth, GN broth
supplemented
with sodium selenite, and SF broth appear to have a better
outcome
with the 6-h culture period than with the 24-h culture period.
In contrast, SBG broth has a better outcome with the 24-h culture
period. To determine the sensitivity for the isolation of
Salmonella spp., artificial specimens were prepared and
adjusted to contain
various numbers of
Salmonella spp., with
a range of 1 to 10
4 CFU. The prepared specimens were then
analyzed by the direct-plating
method and by using GN and SBG
enrichment broths. Both group B
and group D
Salmonella spp.
were included in this examination.
The incubation periods were 6 h
for GN broth and 16 h for SBG
broth. Fifteen repeats were
performed. As shown in Table
4, the
prepared specimens containing 10
4 CFU of
Salmonella spp. appeared to have an isolation rate of
100%
in the direct-plating method, the GN enrichment method, and
the SBG
enrichment method. For specimens containing 10
3 CFU of
group B
Salmonella spp., the isolation rates were 80,
80, and 100% for the direct-plating, GN enrichment, and SBG enrichment
methods, respectively. For specimens containing 10
2 CFU of
group B
Salmonella spp., the isolation rates were 20,
52, and 93% for the direct-plating, GN enrichment, and SBG enrichment
methods, respectively. Furthermore, the positive isolation rates
were
13, 27, and 80% for the direct-plating, GN enrichment, and
SBG
enrichment methods, respectively, when the prepared specimens
contained
10 CFU of group B
Salmonella spp. Thus, the sensitivity
for
isolation of group B
Salmonella spp. is about 100-fold
higher
for SBG broth than for GN broth. The results in Table
4 also
reveal that the sensitivities for isolation of group D and group
B
Salmonella spp. are similar.
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FIG. 1.
Growth of Salmonella on XLD agar plates after
enrichment. Salmonella spp. were incubated in GN broth, SGN
broth, SF broth, and SBG broth. After either 6 or 24 h of
enrichment, the cultures were inoculated onto XLD agar plates. The
salmonellae appear as pink colonies with black centers. The yellow
colonies are the coliforms with the characteristics of
lactose-fermentative organisms.
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TABLE 4.
Sensitivities for recovery of group B and D
Salmonella spp. by the direct-plating XLD, GN enrichment
broth, and SBG enrichment broth methods
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|
We evaluated SBG enrichment broth for the recovery of
Salmonella from feces. With SBG broth as the enrichment, the
isolation
of
Salmonella from clinical specimens was
increased from 89 cases
to 296 cases (Table
1). Thus, the efficacy of
using SBG broth
as the enrichment broth is 3.3-fold higher than that of
using
the direct-plating XLD method alone. A study by Dusch and Altwegg
evaluated the efficacy of enrichment using selenite broth and
tetrathionate broth and found that the sensitivity for the recovery
of
Salmonella strains is higher for tetrationate broth than for
selenite broth. Moreover, the sensitivity in the detection of
Salmonella is increased by using tetrathionate at
concentrations
from 33 to 87% for XLT4 medium and from 34 to 79% for
HE medium
(
2). Thus, the efficacy of using tetrathionate
broth as the
enrichment broth is 2.4-fold higher for the XLT4 method
and 2.3-fold
higher for the HE method in comparison to that of selenite
broth.
Therefore, we conclude that the use of SBG broth as the
enrichment
appears to have a higher efficacy for the recovery of
Salmonella from fecal specimens. The sensitivities of three
enrichment broths,
GN broth, SGN broth, and SBG broth, in detecting
Salmonella strains
in clinical specimens were also compared.
As shown in Tables
2 and
3, the results indicated that the efficacy of
SBG is the
highest among those of the three enrichment broths. Although
the
sodium selenite in SBG has the major inhibitory effect on
coliforms,
the method using GN broth supplemented with sodium selenite
still
has a lower recovery rate of
Salmonella than the SBG
broth method
does. Therefore, other ingredients also contribute some
effects
to the recovery of
Salmonella.
Furthermore, artificial specimens were used in evaluating the recovery
of
Salmonella by using various enrichment broths, including
GN, SGN, SF, and SBG broths. The results in Fig.
1 show that of
these
enrichment broths, the SBG broth has the highest efficacy
for the
recovery of
Salmonella spp. Obviously, this conclusion
allows us to use SBG broth as the enrichment broth and with an
appropriate overnight culture period. This point is also demonstrated
by using clinical specimens, as shown in Table
1. Therefore,
these
results suggest that the optimal culture period and the
sensitivity of
the SBG broth might contribute to the high recovery
rate of
Salmonella. With artificial specimens, the sensitivity
of
SBG broth for the recovery of
Salmonella from feces was also
evaluated. The results shown in Table
4 indicate that the use
of SBG
broth permits isolation of
Salmonella in feces even when
only a small number of bacteria are present. Thus, SBG broth enrichment
allows us to isolate
Salmonella from feces only mildly
infected
with
Salmonella.
In conclusion, our results indicate that SBG broth has high sensitivity
and efficacy for the recovery of
Salmonella from feces.
This
finding has been proven with both clinical specimens and
artificial
specimens. Moreover, this result suggests that SBG
broth is appropriate
to be used in the recovery of
Salmonella in clinical
laboratories.
| |
ACKNOWLEDGMENTS |
We thank Yuan's General Hospital for partial support of the
publication fee.
We also thank Tsan-Zon Liou for discussions and comments on this investigation.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Technology, Fooyin Institute of Technology, Ta-Liao Hsiang,
Kaohsiung Hsien 831, Taiwan, Republic of China. Phone: 886-7-782-7162. Fax: 886-7-782-7162. E-mail: sphuang{at}cc.fy.edu.tw.
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Journal of Clinical Microbiology, December 1999, p. 4120-4123, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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