ferrioxamine e-supplemented pre-enrichment and

8/10/2019 Ferrioxamine E-supplemented Pre-Enrichment And

1/11

International Journal of

Food Microbiology 29 (1996) 81-91

International Journal

of ood Microbiology

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 K och Insti tut e, Wemigerode Branch, Werni gerode, Germany

b Lohmann-Ti erzucht, Veterinar y Laborat ory, Cuxhauen, Germany

Federal Insti tut e of Euregio-I nsti tut e of Research and Development of Envir onmental Technologi es,

Gronau, Germany

Federal Insti tut e of Consumers Healt h Protecti on and Veteri nary M edici ne, Werni gerode Branch

Wemigerode, Germany

Received 20 January 1995; accepted 10 April 1995

Abstract

Supplementation of pre-enrichment broth and enrichment broth media with ferrioxam-

ine E (1 pg/ml) significantly improved the recovery of Salmonella from artificially or

naturally contaminated foods. Based on the selectivity of ferrioxamine E,

Salmonella

enterit idis

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@-Anti-

Salmonella was highly effective.

27 S

typhimut ium 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.

Keyw ords:

Ferrioxamine E ; 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


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82 R. Rersshrodt et al . Int . .I. Food M icrobi ol ogy 29 1996) 81-91

1 Introduction

Isolation and identification of Salmonella whether by traditional cultural meth-

ods or new rapid assay techniques still need enrichment procedure, either non-

selective pre-enrichment or selective enrichments. IS0 and AOAC methods rec-

ommend 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-enrich-

ment media

Salmonella

may be overgrown by competitors. After such pre-enrich-

ment 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. I solat ion of Salmonel l a from art i fi ci all y contami nated egg-whit e albumen)

Preparat i on of contam inat ed 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).


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4/11

84 R. Reissbrodt et al . hr. J. Food M icrobi ol ogy 29 1996) 81-91

2.2. Ferr i oxamine E- supplement

Ferrioxamine E was produced biotechnologically from St rept omyces pi l osus and

kindly provided by Dr. H.H. Peter, Ciba-Geigy, Basel, Switzerland. The concentra-

tion of ferrioxamine E in the supplementation was 1 kg/ml of all the experiments

in this study.

2.3. Resusci t at ion of Salmonel la f rom albumen

Contaminated albumen (strain S. enteritidis 2584/92, phage type [PT] 6 contain-

ing 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-Chrysoidin-

Glycerol-Agar (GCG) plates (SlFIN, Berlin, Germany), incubated at 37C overnight

and read.

2.4. Recoti ery of Salmonel l a enterit i di s 4495/92 by use of di fferent enri chment brot h

media

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. t yphi mur i um of diff erent cel l count s st ored i n al bumen

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.

I solat i on of Salmonel l a from mi xed cult ures

10 ml quantities of albumen were spiked with different mixed cultures contain-

ing very low numbers of

Salmonella

cells and 103-lo4 cells of various competitors

(see Table 1). After pre-enrichment without and with ferrioxamine E-supplementa-

tion 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@-


5/11

R. Reissbrodt et al. ht. J. Food M icr obiology 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. Isol at ion of Salmonel l a from natur all y contami nated meat meal s

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-en-

richment 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. I solat i on of Salmonel la f rom natur all y infect ed chicken gi blet s

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-


6/11

8

R. Rei ssbrodt et al. lnt . J. Food M i crobiol ogy 29 1996) 82-91

Table 2

Isolation of Salmonella from different naturally infected giblets

Number Kind of samples

Isolation of Salmonella after enrichment

of

in Tetrathionate-broth (24 h, 42C)

samples

without ferrioxamine E

with ferrioxamine E

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

not detected

not detected

not detected not detected


not detected

not detected

5 positive S. typhirnur ium 5 positive S. typhimurium

not detected

not detected


not detected

not detected

not detected

1 positive S.

typhzmurium

1 positive S. typhimurium 2 positive S. typhimurium

not detected

not detected

3 positive S. typhirnur ium

3 positive S. typhimurium




not detected

not detected


not detected

not detected


not detected









not detected




1 positive S. typhimurium not detected

not detected


not detected

not detected


not detected

not detected




not detected

not detected

21/X2

33/X2

a Additionally checked in this quality assurance program

ine E also support the recovery of S. enteritidis 4495/92 from BPW and Selenite-

C&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,.


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R. Reissbrodt et al. ht. 1. Food M icrobi ol ogy 29 I 996) 81-91

87

Table 3

Resuscitation of S. enterit idi s 2584/92 from artifically contaminated albumen (approx. 4 cells/ml) after

storage at room temperature and pre-enrichment (37C 6 h) in buffered peptone water with and

without ferrioxamine E

Storage time

Salmonella colonies of 0.1 ml pre-enrichment on GCG-agar

(counts from two plates used)

without supplementation

with ferrioxamine E

3h

Id

4d

6d

11 d

3/o

1000/1000

215

> lOOO/ > 1000

O/O

> 1000/0

13/o > lOOO/ > 1000

13/32

> 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 supple-

mentation. 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@-Anti-

Salmonella. However, after pre-enrichment for 6 h in ferrioxamine E-supple-

mented 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

Kl ebsi el l a pneumoni ae and Citrobacter freundii. In most experiments Dynabeads@-

Anti-Salmonella did concentrate Salmonella cells grown in ferrioxamine E-supple-

mented 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 (approx. 1-2 cells/ml) from different enrichment

nutrient media with and without supplementation, incubation at 37C 6 h. Salmonella colonies on

GCG-agar spreaded with 0.1 ml of each enrichment onto two plates

Supplementation Buffered peptone water Selenite-Cystine broth

Rappaport-Vassiliadis broth

Without

S/O 2/2

6/20

Fe Cl,

O/3

O/O

40/40

3 Ccg/mt

Ferrioxamine E > lOOO/ > 1000 > loo/ > 100 _ lOO/ > 100

1 pg/mf


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R. Rei ssbrodt et al. ht. J. Food Mi crobiol ogy 29 1996) 81-91

89

counts of Salmonella and comparing the experiments with E. coli and Proteus

mirabi i is 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 multipli-

cation 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. enteri-

t id is seeded (without any of competitors) in raw eggs based on growth promotion

using ferrous sulphate together with immunomagnetic separation employing Dy-

nabeads@-Anti-Salmonella has been described (Cudjoe et al., 1994). The applica-

tion 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 M organell a

group have an uptake and utilization system of ferrioxamine E. Therefore, this

iron source is selective with respect to these competitors. However, ferrioxamine E


10/11

90 R. Reissbrodt et al. /ht. J. Food Microbiology 29 1996) 81-91

can be used by Ci t robacter spp., K l ebsiell a spp., Pseudomonas spp. Enterobacter

SPPY

and Yersinia enterocolitica. Therefore, while isolation and detection of

Salmonella should be facilitated by supplementation of pre-enrichment or enrich-

ment 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 incuba-

tion 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-enrich-

ment 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 effec-

tive 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 pro-

posed 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-pro-

ducing

Citrobacter

spp. (Miller et al.,

19911, although non-FeS-producing

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

Salmonell a-E. coli -Prot eus mi rabil is

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. pneumoni ae and C. freundii.

The function of ferrioxamine E as a growth factor of Salmonella in Tetrathion-

ate 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-supple-

mented 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


11/11

R. Reissbrodt t al. ht. J. Food Mi crobiol ogy 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.

cknowledgement

We are very grateful to Dr. Derwent Swaine, Oxford, England, for critical

reading of the manuscript.

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