International

Journal

of Food Microbiology

International Journal of
Food Microbiology 29 (1996) 81-91

Ferrioxamine E-supplemented pre-enrichment and

enrichment media improve various isolation methods for
Salmonella
R. Reissbrodt

a3*, E. Vielitz b, E. Kormann , W. Rabsch d, H. Kiihn a

a Robert Koch Institute, Wemigerode Branch, Wernigerode, Germany

b Lohmann-Tierzucht, Veterinary Laboratory, Cuxhauen, Germany
Federal Institute of Euregio-Institute of Research and Development of Environmental Technologies,
Gronau, Germany
Federal Institute of Consumers Health Protection and Veterinary Medicine, Wernigerode Branch
Wemigerode, Germany

Received 20 January 1995; accepted 10 April 1995

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

R. Rersshrodt et al. / Int. .I. Food Microbiology 29 (1996) 81-91

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

2.1. Isolation of Salmonella from artificially contaminated egg-white (albumen)

Preparation of contaminated albumen: Eggs less than 2 days old were purchased
from a local producer and stored for no more than 2 days before use. Egg shells
were wiped with ethanol (70% v/v), cracked and the albumen was collected. The
albumen
was artificially
contaminated
at a level of l-10
Salmonella cells/ml
(strains of S. enteritidis and S. typhimurium) and in the case of mixtures with the
contaminants
as listed in Table 1. All bacteria tested in experiments
with albumen
were taken from the collection of the Robert Koch Institute, Wernigerode
Branch,
Germany.
The contaminated
albumen was stored overnight at room temperature.
Before inoculation,
the cell counts of the inoculum were determined
by the most
probable number technique
(MPN).

XLD

XLT-4

Selective
nutrient
media

directly
with Dynabeads
directly
with Dynabeadsm
directly
with Dynabeads

1.4 X lo4 E. coli

8 X lo3 P. mirabilis
2.0~ lo4 P. aeruginosa

20 S. iyphimwium 340/94

1 S. fyphimurium 340/94

1 5. typhimurium 340/94

17 S. enteritidis 2584/92

1.4 X lo4 E. coli

2.0X lo4 P. aeruginosa
1.4X lo4 E. coli

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

Plated onto selective

nutrient medium

Cells of the competitors

spiked with

Salmonella cells

10 ml albumen

(with and without

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

E) by use of either XLD-

with ferrioxamine

ferrioxamine

Colony counts after pre-enrichment

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. /hr.

J. Food Microbiology 29 (1996) 81-91

2.2. Ferrioxamine E-supplement

Ferrioxamine
E was produced biotechnologically
from Streptomyces pilosus and
kindly provided by Dr. H.H. Peter, Ciba-Geigy,
Basel, Switzerland.
The concentration of ferrioxamine
E in the supplementation
was 1 kg/ml
of all the experiments
in this study.
2.3. Resuscitation of Salmonella from albumen
Contaminated
albumen (strain S. enteritidis 2584/92, phage type [PT] 6 containing the 37 MDa plasmid) was stored at room temperature
until 11 days. At
different
intervals
1 ml of this suspension
was diluted with 9 ml of buffered
peptone water (BPW, Oxoid CM 509, Unipath Ltd., Basingstoke,
England; without
and with ferrioxamine
E-supplementation),
cultivated
at 37C by shaking for 6 h.
0.1 ml of the pre-enrichment
cultures
were streaked
onto Galle-ChrysoidinGlycerol-Agar
(GCG) plates (SlFIN, Berlin, Germany), incubated at 37C overnight
and read.
2.4. Recotiery of Salmonella enteritidis 4495/92
media

by use of different enrichment broth

A few cells of S. enteritidis 4495/92 (PT 4) (approx. l-2/ml)

in albumen were
diIuted in a ratio of 1:lO with BPW (Oxoid CM 509), Selenite-Cystine
broth (Oxoid
CM 699) or Rappaport-Vassiliadis
broth (Oxoid, CM 6691, respectively.
The
pre-enrichment
broth and both the enrichment
broth media were used without
supplementation
or supplemented
with FeCl, (3 pg/ml)
or with ferrioxamine
E.
After shaking at 37C for 6 h, 0.1 ml of each suspension
was spread onto GCG
plates (two for each experiment),
incubated
at 37C and examined.
2.5. Recovery of S. typhimurium of different cell counts stored in albumen
Less than 10 to ca. 1.4 X lo3 S. typhimurium 340/94 (PT 2/m) cells in 10 ml
albumen were treated as described above and incubated
at 37C in BPW (Oxoid
CM 509) without and with ferrioxamine
E-supplementation
with shaking. Aliquots
were removed after 4,5 and 6 h, diluted and 0.1 ml of the dilutions spread onto
GCG-plates.
The colony counts were monitored
after incubation
at 37C overnight.
2.6. Isolation of Salmonella from mixed cultures
10 ml quantities
of albumen were spiked with different mixed cultures containing very low numbers of Salmonella cells and 103-lo4 cells of various competitors
(see Table 1). After pre-enrichment
without and with ferrioxamine
E-supplementation by shaking at 37C for 6 h, 0.1 ml volumes of the suspensions
were spread onto
XLD-agar (Oxoid CM 469) and XLT-6agar
(Difco, Code-No. 0234-17-9, Detroit,
USA). In addition,
1 ml of the pre-enrichments
were treated with Dynabeads@-

R. Reissbrodt

et al. /ht.

J. Food Microbiology

29 (1996) 81-91

85

Anti-Salmonella (kindly provided by Dynal, Oslo, Norway). Concentration and

isolation of Salmonella were performed according to the instructions from the
manufacturers. The Dynabeads@-Anti-Salmonella
concentrates were diluted and
distributed onto XLD- and XLT4-agar. The selective agar media were incubated
at 37C overnight and the cell counts monitored.
2.7. Isolation of Salmonella from naturally contaminated meat meals

50 samples of different meat meals were checked in this study by (1) a

conventional method using a pre-enrichment and two selective enrichment media
and (2) by application of ferrioxamine E-supplemented BPW.
Each sample was evenly divided and placed at a 1:lO ratio (25 g/225 ml) in
BPW (E. Merck, Darmstadt, Germany, Art.-Nr. 7228). Using the conventional
method the suspensions in BPW were incubated at 37C for 24 h. These pre-enrichment cultures were inoculated into the selective enrichment broth media
Tetrathionate
broth (Art.-Nr. 10863) and also in Rappaport-Vassiliadis-broth,
Art.-Nr. 7770 (both purchased from E. Merck, Darmstadt, Germany); (5 ml of
pre-enrichment
culture were diluted with 50 ml of Tetrathionate
broth and
incubated at 37C for 24 b; 0,l ml pre-enrichment culture were diluted with 10 ml
of Rappaport-Vassiliadis-broth
and incubated at 42,9C for 20-24 h). These
enrichment broth cultures were plated onto XLD-agar (Oxoid, CM 469) and
Rambach-agar (E. Merck, Darmstadt, Germany, Art.-No. 7500). Both selective
Salmonella-agar media were incubated at 37C overnight and examined for typical
Salmonella colonies.
The suspensions in ferrioxamine E-supplemented BPW were incubated at 37C
by shaking for 6 and 24 h. Subsequently, 0.1 ml of these pre-enrichment cultures
were directly spread onto XLD- and Rambach-agar as already described before,
incubated at 37C overnight and examined. Typical Salmonella colonies were
checked serologically and confirmed by conventional biochemical tests.
2.8. Isolation of Salmonella from naturally infected chicken giblets
762 different fresh samples obtained from a poultry farm (see Table 2) were
directly enriched in Tetrathionate broth (Oxoid, CM 29) in a ratio of 1:lO. Each
sample was enriched without and with supplementation with ferrioxamine E at
42C for 24 h. Subsequently, a loop of that enrichment cultures was streaked onto
Brilliant Green agar (Oxoid, CM 329) and incubated at 37C for 24 h. The
Salmonella colonies grown were checked as described above.

3. Results

Resuscitation from low numbers of S. enteritidis 2584/92 cells after storage in

albumen was extensively supported by ferrioxamine E (Table 3) for up to 11 days.
Supplementation of various pre-enrichment and enrichment media with ferrioxam-

R. Reissbrodt et al. /lnt. J. Food Microbiology 29 (1996) 82-91

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

Isolation of Salmonella after enrichment

in Tetrathionate-broth
(24 h, 42C)

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,.

R. Reissbrodt et al. /ht.

1. Food Microbiology 29 (I 996) 81-91

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

Salmonella colonies of 0.1 ml pre-enrichment

(counts from two plates used)

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

> lOOO/ > 1000

> 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

_ lOO/ > 100

broth

Salmonella

0
0
0
18

Cells inoculated into

10 ml of albumen

< 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

R. Reissbrodt et al. /ht.

J. Food Microbiology 29 (1996) 81-91

89

counts of Salmonella and comparing

the experiments
with E. coli and Proteus
mirabiiis in the mixture no growth of these competitors
was seen on XLT-Cagar,
but there was growth on XLD-agar (rsults not shown).
From the 50 samples of meat meals no Salmonella were detected by use of a
conventional
method (results not shown). However, from two of these 50 samples
Salmonella colonies were detected on XLD- and Rambach-agar
after direct plating
of a 6 h culture in ferrioxamine
E-supplemented
BPW. A further sample was
positive on both of the selective agar media plated after 24 h shaking at 37C in
ferrioxamine
E-supplemented
BPW.
S. typhimurium was isolated from 27 of 762 naturally infected giblets by direct
enrichment
in Tetrathionate
broth. However, 33 isolations of S. typhimurium were
made with the ferrioxamine
E-supplemented
Tetrathionate
broth. Positive isolates
were often obtained from liver and intestine, and also from ovary samples (Table 2.

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

can be used by Citrobacter spp., Klebsiella spp., Pseudomonas spp. Enterobacter

while isolation
and detection
of
SPPY and Yersinia enterocolitica. Therefore,
Salmonella should be facilitated by supplementation
of pre-enrichment
or enrichment media with ferrioxamine
E, the additional
selective step, by culture or by
newer rapid methods,
should especially
consider
the need to suppress
these
competitors.
In this study we have shown the effectiveness
of ferrioxamine
E in resuscitation
(Table 3) and recovery of S. enteritidis and S. typhimurium ( Tables 4 and 5) under
different
conditions.
Isolation
of Salmonella was possible from a few cells with
short incubation
times in ferrioxamine
E-supplemented
BPW or Selenite-Cystine
broth. Shaking for only 6 h enabled
detectable
numbers
of Salmonella to be
achieved. Using Rappaport-Vassiliadis
medium the effect was less because this
medium contains more available iron than other Salmonella enrichment
media.
The effectiveness
of ferrioxamine
E-supplementation
in a shorter
Salmonella
isolation
time depends
also on the levels of Salmonella in the contaminated
samples. Few S. typhimurium cells could be detected with the short time incubation in supplemented
BPW only, but higher initial cell counts were detectable
in
the shorter time (Table 5).
Ferrioxamine
E significantly
improved isolation of low numbers of Salmonella
from mixed cultures when used as a supplement
in BPW. Salmonella colonies
could be detected on XLD- or XLT-4 agars by direct plating after 6 h pre-enrichment from mixtures in a ratio of approx. 1 Salmonella cell (S. enteritidis or S.
typhimurium) to lo-lo4
cells of the competitors.
An immunocapturing
method
with the immunomagnetic
Dynabeads=-Anti-Salmonella
was shown to be an effective method (Mansfield
and Forsythe,
1993) and its effectiveness
was further
enhanced
by ferrioxamine
E-supplementation
of the pre-enrichment
medium,
Thus highly selective Dynabeads-Anti-Salmonella
in combination
with the proposed pre-enrichment
should significantly
improve the isolation rate of Salmonella.
XLD- and XLT-4 agars were not significantly
different
in recovery rate. An
advantage of XLT-4 agar may be the inhibition
of Proteus-growth
and of FeS-pronon-FeS-producing
ducing
Citrobacter spp. (Miller
et al., 19911, although
Salmonella are difficult to detect and lower cell counts of Salmonella were seen
compared with results on XLD-agar.
The selective function of ferrioxamine
E on
Salmonella in a Salmonella-E. coli-Proteus mirabilis mixture was very easy to
demonstrate.
Nevertheless,
Salmonella could be detected
also from the other
mixtures used except for the experiment
of direct plating on XLT-4-agar
from the
mixture with K. pneumoniae and C. freundii.
The function of ferrioxamine
E as a growth factor of Salmonella in Tetrathionate broth (Table 2) seems to be an important result allowing use of this enrichment
broth in the isolation of Salmonella from different
materials
contaminated
with
Proteus spp. These organisms also possess the enzyme tetrathionate
reductase and
can survive in this selective broth. The combination
of ferrioxamine
E-supplemented Tetrathionate
broth and subsequent
use of XLD-agar may be a successful
method in isolation of Salmonella. Supplementation
of Tetrathionate
broth with
ferrioxamine
E resulted
in higher isolation
rates (18.2% more than without

R. Reissbrodtet 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

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