Documente Academic
Documente Profesional
Documente Cultură
Journal
of Food Microbiology
International Journal of
Food Microbiology 29 (1996) 81-91
Abstract
Supplementation
of pre-enrichment
broth and enrichment
broth media with ferrioxamine E (1 pg/ml)
significantly
improved
the recovery of Salmonella
from artificially or
naturally
contaminated
foods. Based on the selectivity
of ferrioxamine
E, Salmonella
enteritidis and S. typhimurium
could be isolated also from various mixed cultures (one
Sulmonella cell in 103-104-fold
concentration
of cells of competitors)
by shaking for 6 h in
supplemented
buffered peptone water followed by cultivation on XLD- or XLT-4 agars.
Isolation of Salmonella from these pre-enrichment
cultures by use of Dynabeads@-AntiSalmonella was highly effective. 27 S. typhimutium strains were isolated from 762 naturally
infected chicken giblets by use of unsupplemented
Tetrathionate
broth. However, 33 S.
typhimurium
isolates were obtained with ferrioxamine
E-supplemented
Tetrathionate
broth
from the same samples. Three Salmonelk
isolates out of 50 evenly divided meat meal
samples were obtained
by use of ferrioxamine
E-supplemented
buffered
peptone water
followed by direct streaking onto XLD- and Rambach agars, no Salmonella isolates could
be detected by the conventional
method.
Keywords:
Ferrioxamine
; Supplemented
pre-enrichment
and
enrichment
media;
Salmonella
* Corresponding author. Wernigerode Branch, Burgstrape 37, D-38855 Wernigerode, Germany. Tel.:
03943/679-O. Fax: 03943/679 207.
0168-1605/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved
SSDIO168-1605(95)00024-O
82
1. Introduction
Isolation and identification
of Salmonella whether by traditional
cultural methods or new rapid assay techniques
still need enrichment
procedure,
either nonselective pre-enrichment
or selective enrichments.
IS0 and AOAC methods recommend both kinds of enrichments
which are laborious and time-consuming.
The
use of a pre-enrichment
step is an agreed essential stage for isolation of Salmonella
from food, feed, industrial
processing
or environmental
samples. Questions
arise
concerning
increased sensitivity and short incubation
times when isolation of small
numbers
of Salmonella are being considered.
The time for enrichments
to yield
around 104-10h cells/ml
is the measure of the effectiveness.
Short pre-enrichment
times of 6 h would be necessary to obtain a one day
cultural detection method or in a more rapid detection by different rapid assays. A
6 h pre-enrichment
can only be considered
if it is a safe procedure (DAoust et al.,
1990; Tate et al., 1990; Humbert and Colin, 1991). Using non-selective
pre-enrichment media Salmonella may be overgrown by competitors.
After such pre-enrichment stages, a selective detection method is then required.
The natural siderophores
Ferrioxamine
E or G act as more or less selective
growth factors of Salmonella (Reissbrodt
and Rabsch, 1993). Most Salmonella
serotypes associated with in food poisoning incidents tested grew from a few cells
to detectable
numbers in 6 h by use of buffered peptone water supplemented
with
these siderophores.
This paper reports on the effectiveness of ferrioxamine
E-supplemented
buffered
peptone water as a pre-enrichment
system and of ferrioxamine
E-supplemented
enrichment
broth media in the isolation of non-typhoid
Salmonella from foods,
both artificially or naturally
infected. Three selective nutrient
agar media (XLD,
Rambach
and XLT-4 agars) were checked after these enrichment
procedures.
Dynabeads-Anti-Salmonella
were also used in immunocapturing
experiments
to
isolate Salmonella from mixed cultures.
2. Material
and methods
XLD
XLT-4
Selective
nutrient
media
directly
with Dynabeads
directly
with Dynabeadsm
directly
with Dynabeads
20 S. iyphimwium 340/94
1 S. fyphimurium 340/94
1 5. typhimurium 340/94
17 S. enteritidis 2584/92
coli
mirahilis
coli
mirabilis
5X103
4 X lo3
5 X lo3
4 x 10
5 S. enteritidis 2584/92
E.
P.
E.
P.
directly
with Dynabeads
directly
with Dynabeads
directly
with Dynabeads
5 X 10 a K. pneumoniae
9 X 10 3 C. freundii
11 S. enteritidis 2584/92
spiked with
Salmonella cells
10 ml albumen
0
0
0
102
2x102
5x102
0
4x IO2
0
0
0
0
4x102
2x10*
2x102
ca. 10
80
2x102
10
3x104
0
0
0
0
non-Salmonella
supplementation
Salmonella
without
20
1,l x 10
5x102
10
IO3
5x103
0
5x103
2x102
102
5x102
6~10~
Salmonella
2x102
3x102
3x102
ca. 10
5x102
3x103
ca. 10
8~10~
0
0
0
0
non-Salmonella
with ferrioxamine
ferrioxamine
Table 1
Isolation of low numbers of Salmonella cells in mixed cultures from albumen after pre-enrichment
or XLT-4-agar and immunomagnetic
separation
(IMS, DynabeadsO-Anti-Salmonella)
co
84
R. Reissbrodt
et al. /ht.
J. Food Microbiology
29 (1996) 81-91
85
3. Results
86
Table 2
Isolation
of Salmonella
from different
naturally
Kind of samples
72
3
8
61
6
75
5
38
4
6
58
3
26
6
12
48
3
39
3
10
10
10
83
2
6
4
6
2
8
5
64
10
4
20
14
28
762
ovary, intestine
organs, intestine of turkeys a
organs, brain
ovary, intestine
liver, intestine
ovary, intestine
organs, ovary, brain, intestine
ovary, intestine
liver, intestine
liver, intestine
ovary, intestine
organs, egg yolk, intestine
ovary, intestine
liver, intestine
liver, intestine
ovary, intestine
heart, liver, spleen
ovary, intestine
organs
ovary, intestine
liver, intestine
liver, intestine
ovary, intestine
windpipe, intestine
ovary, intestine
ovary, intestine
ovary, intestine
ovary, intestine
ovary, intestine
intestine
ovary, intestine
ovary, intestine, liver
organs, intestine of mice
liver, intestine
liver, intestine
ovary, intestine
without
checked
in this quality
giblets
Number
of
samples
a Additionally
infected
ferrioxamine
not detected
not detected
not detected
not detected
5 positive S.
not detected
not detected
not detected
not detected
1 positive S.
not detected
3 positive S.
not detected
2 positive S.
1 positive S.
not detected
not detected
not detected
not detected
not detected
3 positive S.
4 positive S.
not detected
not detected
2 positive S.
not detected
1 positive S.
1 positive S.
not detected
not detected
not detected
not detected
not detected
not detected
4 positive S.
not detected
21/X2
assurance
typhirnurium
typhimurium
typhirnurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
with ferrioxamine
not detected
not detected
not detected
not detected
5 positive S.
not detected
not detected
not detected
1 positive S.
2 positive S.
not detected
3 positive S.
not detected
2 positive S.
1 positive S.
not detected
not detected
not detected
not detected
1 positive S.
4 positive S.
4 positive S.
not detected
not detected
2 positive S.
2 positive S.
1 positive S.
not detected
1 positive S.
not detected
not detected
not detected
not detected
not detected
4 positive S.
not detected
33/X2
typhimurium
typhzmurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
typhimurium
program
ine E also support the recovery of S. enteritidis 4495/92 from BPW and SeleniteC&tine-broth.
Supplementation
with FeCl, was without
any effect (Table 4).
Ferrioxamine
E also acts in a positive way on the recovery of this strain from
Rappaport-Vassiliadis
broth. A positive effect was also seen in this instance by
supplementation
with FeCl,.
Table 3
Resuscitation
of S. enteritidis 2584/92 from artifically contaminated
albumen
storage at room temperature
and pre-enrichment
(37C 6 h) in buffered
without ferrioxamine
E
Storage
time
without
supplementation
>
>
>
>
O/O
13/o
13/32
4 cells/ml)
after
water with and
on GCG-agar
with ferrioxamine
3/o
215
3h
Id
4d
6d
11 d
(approx.
peptone
87
1000/1000
lOOO/ > 1000
1000/0
lOOO/ > 1000
lOOO/ > 1000
Supplementation
of BPW supports multiplication
of S. typhimutium 340/94 in a
range starting from a few cells up to 1.4 X lo3 cells/l0
ml albumen
(Table 5).
After 4 and 5 h incubation
with shaking cells could not detected without supplementation.
However, detectable
counts of S. typhimurium 340/94 were obtained
after these times by using ferrioxamine
E-supplemented
BPW (Table 5). From ca.
140 cells/l0
ml of albumen
2 X lo5 cells/ml
were produced
by pre-enrichment
with ferrioxamine
E-supplemented
BPW after 6 h incubation
at 37C with shaking.
Isolation of low numbers of Salmonellu cells in mixed cultures was difficult after
pre-enrichment
in BPW without
ferrioxamine
E-supplementation
(Table
1).
Salmonellu colonies were detected
on one occasion only by direct plating on
XLD-agar
and on three occasions
by the additional
use of Dynabeads@-AntiSalmonella.
However,
after pre-enrichment
for 6 h in ferrioxamine
E-supplemented BPW Salmonella colonies could be detected in all experiments
except with
the direct plating method on XLT-4 from a mixture of S. enteritidis 2584/92 with
Klebsiella pneumoniae and Citrobacter freundii. In most experiments
Dynabeads@Anti-Salmonella
did concentrate
Salmonella cells grown in ferrioxamine
E-supplemented BPW. Here, higher cell counts were monitored on XLT-4- and XLD-agars.
Dyna-beads@-Anti-Salmonella
did not exclude the competitors
used in the mixed
cultures. Both selective agar media showed only small differences in the cell counts
of Salmonella detected. More competitors
were seen on XLD-agar, but also higher
Table 4
Recovery of S. enteritidis 4495/92 from albumen
nutrient
media with and without supplementation,
GCG-agar
spreaded with 0.1 ml of each enrichment
Supplementation
Buffered
Without
Fe Cl,
S/O
2/2
O/3
O/O
> loo/
3 Ccg/mt
Ferrioxamine
1 pg/mf
peptone
water
(approx.
1-2 cells/ml)
from different
enrichment
incubation
at 37C 6 h. Salmonella colonies on
onto two plates
Selenite-Cystine
broth
Rappaport-Vassiliadis
6/20
40/40
> 100
broth
Salmonella
0
0
0
18
< 10
ca. 14
ca. 140
ca. 1.400
0
13
120
1.400
without
supplementation
4h
10 to 1.4.10
340/94
(approx.
of S. ryphimurium
Cells detected in
0.1 ml of the
inoculated albumen
after 18 h
Table 5
Detection
8
27
> 200
ca. 3.000
with
ferrioxamine E
after different
5h
0
56
390
17.100
without
supplementation
49
66
1.340
22.000
after
7
- 400
ca. 1.000
ca. 33.000
without
supplementation
6h
times of pre-enrichment
with
ferrioxamine E
culture
incubation
in 0.1 ml of pre-enrichment
ml albumen)
cells detected
cells/l0
ca.
ca.
ca.
ca.
200
600
20.000
200.000
with
ferrioxamine E
culture
89
4. Discussion
Almost all living cells, including
microorganims,
require iron as an essential
element. Because iron may not be in a readily available form in most samples or in
nutrient culture media, addition of an effective iron source may improve multiplication of bacteria. Iron in the form of ferrous sulphate has been demonstrated
to
promote the growth of Gram-negative
bacteria in eggs (Garibaldi,
1960; Clay and
Board, 1991) and its successful use at levels of 35 mg/l in a non-selective
broth to
isolate Salmonella from raw eggs has been reported (Gast and Beard, 1992; Gast,
1993). The addition
of ferrous sulphate
to saturate
ovotransferrin
significantly
enhanced
the growth of, for example,
S. enteritidis in raw eggs compared
to
unsupplemented
samples. A protocol for the isolation and detection
of S. enteritidis seeded (without any of competitors)
in raw eggs based on growth promotion
using ferrous sulphate together with immunomagnetic
separation
employing
Dynabeads@-Anti-Salmonella
has been described (Cudjoe et al., 1994). The application of this protocol enables definitive detection of S. enteritidis from eggs within
30 h. Such iron salts (ferrous sulphate, ferriammonium
citrate) also promote the
growth of competetive
bacteria.
Separation
of Salmonella from mixed cultures
needs highly selective methods. Supplementation
of nutrient
media with an iron
source for more or less specific use by pathogenic
bacteria could be advantageous
for improved isolation,
quantitatively
as well as qualitatively,
Ferrioxamine
E, a
trihydroxamate
siderophore,
at a level of not more than 1 mg/l broth culture
promotes
the growth of all of the Salmonella serovars tested (Reissbrodt
and
Rabsch,
1993) and improves
extensively
the motility of Salmonella on motility
semisolid media (e.g. MSRV, Oxoid CM910 or DIASSALM,
LAB 537, LABM,
Bury, England;
Pless and Reissbrodt,
1995). Ferrioxamine
E supplies
iron to
Salmonella by a special uptake and utilization
system of the cell, not by saturation
of ovotransferrin.
No strains of E. coli, the Proteus-Providencia- and Morganella
group have an uptake and utilization
system of ferrioxamine
E. Therefore,
this
iron source is selective with respect to these competitors.
However, ferrioxamine
E
90
R. Reissbrodt
et al. /ht.
J. Food Microbiology
29 (1996) 81-91
91
supplementation)
from naturally infected giblets. The detection of three SuZmoneZla
isolates out of 50 meat meal samples examined using ferrioxamine
E-supplemented
BPW compared
to no isolations
using the conventional
method underlines
the
effectiveness
of this growth factor. Shaking of the supplemented
pre-enrichment
culture at 37C will have contributed
to the rapid isolation of Salmonella avoiding
a further enrichment
step.
The higher isolation rates seen with the experiments
on giblets and meat meal,
where the iron limitation
is not as severe as in the case of albumen,
shows that
supplementation
with ferrioxamine
E is likely to be of general benefit.
Acknowledgement
We are very grateful
reading of the manuscript.
to Dr.
Derwent
Swaine,
Oxford,
England,
for critical
References
Clay, C.E. and Board, R.G. (1991) Growth of Salmonella enteritidis in artificially contaminated
hens
shells eggs. Epidemiol.
Inf. 106, 271-281.
Cudjoe, KS., Krona, R., Gron, B. and Olsen, E. (19941 Use of ferrous sulphate and immunomagnetic
separation
to recover Salmonella enteritidis from raw eggs. Int. J. Food Microbial. 23, 149-158.
DAoust, J.D., Sewell, A. and Jean, A. (1990) Limited sensitivity of short (6 h) selective enrichment
for
detection of foodborne
Salmonella.
J. Food Prot. 53, 562-565.
Garibaldi,
J.A. (19601 Factors in egg white which control growth of bacteria. Food Res. 25, 337-344.
Cast, R.K. (1993) Recovery of Salmonella enteritidis from inoculated
pools of egg contents.
J. Food
Prot. 56, 21-24.
Cast, R.K. and Beard, C.W. (1992) Detection
and enumeration
of Salmonella enteritidis in fresh and
stored eggs laid by experimentally
infected hens. J. Food Prot. 55, 152-156.
Humbert,
F. and Colin, P. (19911 Methods for isolating and identifying
Salmonella.
Proceedings
of a
meeting held at the Huhn und Schwein exhibition, Hannover,
28th June.
Mansfield, L.P. and Forsythe, S.J. (1993) Immunomagnetic
separation
as an alternative
to enrichment
broth for Salmonella
detection. Lett. Appl. Microbial.
16, 122-125.
Miller, R.G., Tate, CR., Mallinson,
E.T. and Scherrer,
J.A. (19911 Xylose-Lysine-Tergitol
4: An
improved selective agar medium for the isolation of Salmonella.
Poultry Sci. 70, 2429-2432.
Pless, P. and Reissbrodt,
R. (19951 Improvement
of Salmonella detection on motility-enrichment
media
by ferrioxamine
E supplementation
of pre-enrichment
culture. Int. J. Food Microbial. 27, 147-159.
Reissbrodt,
R. and Rabsch, W. (1993) Selective pre-enrichment
of Salmonella from eggs by siderophore
supplements.
Zbl. Bakt. 279, 344-353.
Tate, C.R., Miller, R.G., Mallinson, E.T. and Douglass, L.W. (1990) The isolation of Salmonella
from
poultry environmental
samples by several enrichment
procedures
using plating media with and
without novobiocin.
Poultry Sci. 69, 721-726.