Hus Suwa

Journal of Applied Microbiology 2004
doi:10.1111/j.1365-2672.2004.02450.x
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Molecular characterization, technological properties and

safety aspects of enterococci from Hussuwa, an African
fermented sorghum product
N.M.K. Yousif1, P. Dawyndt2, H. Abriouel1, A. Wijaya1, U. Schillinger1, M. Vancanneyt2, J. Swings2,

H.A. Dirar3, W.H. Holzapfel1 and C.M.A.P. Franz1
Federal Research Centre for Nutrition, Institute of Hygiene and Toxicology, Karlsruhe, Germany, 2BCCM/LMG Bacteria Collection,
Laboratory of Microbiology, University of Ghent, Ghent, Belgium, and 3Department of Biotechnology, Faculty of Agriculture, Shambat
University of Khartoum, Khartoum, Sudan
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2003/1196: received 30 December 2003, revised and accepted 9 August 2004
ABSTRACT
N.M.K. YOUSIF, P. DAWYNDT, H. ABRIOUEL, A. WIJAYA, U. SCHILLINGER, M. VANCANNEYT, J. SWINGS,

H . A . D I R A R , W . H . H O L Z A P F E L A N D C . M . A . P . F R A N Z . 2004.
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Aims: To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their
technological properties and safety for possible inclusion in a starter culture preparation.
Methods and Results: Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by
using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment
length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were
not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful
technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None
produced a-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics
ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin,
erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence
determinants occurred in some strains.
Conclusions: Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains
exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible
sugars, which may aid in stabilizing and improving the digestibility of the product respectively.
Significance and Impact of the Study: Enterococci were shown to play a role in the fermentation of African
foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a
hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.
Keywords: enterococci, fermented foods, genotyping, Hussuwa.
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INTRODUCTION
Sorghum is a drought-tolerant plant, grown in the semiarid tropical regions of Africa and Asia (Dogett 1988).
Many indigenous fermented foods from Sudan are based
Correspondence to: Charles M.A.P. Franz, Federal Research Centre for Nutrition,
Institute for Biotechnology and Molecular Biology, Haid-und-Neu-Strasse 9,
D-76131 Karlsruhe, Germany (e-mail: charles.Franz@bfe.uni-karlsruhe.de).
on sorghum fermentation (Dirar 1993); among these,

Hussuwa is a semi-solid, dough-like food, which, so far
has not been subjected to microbiological investigation.
Traditionally, Hussuwa production relies on spontaneous
fermentation by the autochthonous microflora. It is
produced from a semi-solid paste of sorghum flour and
sorghum malt in a 1 : 05 ratio. The paste is left to
ferment for 12 h, after which it is cooked lightly in the
2004 The Society for Applied Microbiology
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Dispatch: 5.10.04
Author Received:
Journal: JAM CE: Nithya

No. of pages: 13 PE: Raymond
2 N . M . K . Y O U S I F ET AL.
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about the involvement of enterococci with African fermented foods, this study not only aimed to characterize the
enterococci isolates, but also to investigate some possible
technologically relevant traits and safety aspects of these
isolates. These investigations, thus, attempted to evaluate
whether these enterococci may play a functional role in
fermentation and whether these bacteria should be taken
into consideration when selecting a starter culture preparation for the production of Hussuwa.
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MATERIALS AND METHODS

Microbiological sampling
Samples were taken from 11 different stages during two

different Hussuwa fermentations carried out in different
surroundings. At each sampling point, a 50-g sample was
placed in a sterile blender jar and blended for 30 s, after
which, serial 10-fold dilutions were made in quarterstrength Ringers solution (Merck, Darmstadt, Germany).
One hundred microlitres of suitable dilutions were spread
plated onto de Man, Rogosa and Sharpe (MRS) agar
(Merck) to determine the LAB count and to isolate bacteria
associated with fermentation. MRS agar plates were incubated anaerobically at 37C for 3 days, and 10 colonies from
the plates of the highest dilution were picked at random for
characterization. Before characterization, cultures were
checked for purity by streak plating and phase contrast
microscopy. Stock cultures were kept at )80C in MRS
broth with 15% glycerol added.
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form of dough or as pancakes. After cooling, a further half

portion of sorghum malt is kneaded into the dough. This
is then either left to ferment for a further 2448 h and
cooked on a hot plate until all moisture is expelled or
formed into fist-sized balls and placed in an earthenware
pot Zeer. The Zeer is covered, sealed with mud and
buried under the fireplace for up to 2 months. The
cooking of three daily meals over it ensures a continuous
warming throughout this period of fermentation. Both
lactic and ethanolic fermentations take place during this
process, which, finally results in a sweetsour product
(Dirar 1993). Similar to many other African fermented
foods, Hussuwa is prepared in a traditional way and often
on a small-scale, home-production level. As a result, such
indigenous foods often suffer from problems such as
inconsistent quality, hygienic risks and short storage life
(Onyekwere et al. 1989).
Enterococci are lactic acid bacteria (LAB) that occur in a
wide range of habitats including the gastrointestinal tract of
animals, soil, surface waters and on plants (Franz et al.
1999a). They are also associated with a number of European
fermented foods such as dry-fermented sausages or traditional cheeses produced in Mediterranean countries from
pasteurised or raw milk (Ordonez et al. 1978; LitopoulouTzanetaki 1990; Cogan et al. 1997; Franz et al. 1999a). In
addition, the enterococci, especially Enterococcus faecalis
strains, have also been associated with African fermented
sorghum foods (Mohammed et al. 1991; Hamad et al. 1997).
In cheeses, enterococci have been suggested to play an
important role in ripening as well as flavour and aroma
development (Ordonez et al. 1978; Trovatelli and Schiesser
1987; Centeno et al. 1996; Cogan et al. 1997), while the role
of enterococci in vegetable-based African fermented foods
remains largely unknown.
Some strains of enterococci are opportunistic human
pathogens and can cause nosocomial infections such as
bacteraemia, endocarditis, urinary tract and other infections
(Murray 1990; Morrison et al. 1997). A contributing factor
to pathogenesis of enterococci is their resistance to a wide
variety of antibiotics including the glycopeptide antibiotic
vancomycin (Murray 1990; Landman and Quale 1997;
Leclercq 1997). This, in combination with the production of
virulence factors such as aggregation substance (AS),
gelatinase (Gel), cytolysin (Cyl), enterococcal surface protein (Esp), adhesin to collagen from E. faecalis (Ace) and
Enterococcus endocarditis antigen (Efafs or Efafm from E.
faecalis or E. faecium respectively), determines that enterococci are not considered as generally recognised as safe
(GRAS) organisms.
This study aimed to characterize the enterococci associated with the Hussuwa production, as these bacteria
constituted a relevant proportion (ca10%) of LAB isolated
from this product during fermentation. As little is known
Phenotypic characterization
All Gram-positive, catalase-negative bacteria isolated from
MRS agar were considered as presumptive LAB. Presumptive LAB strains were further characterized by testing
production of gas (CO2) from glucose, growth in MRS broth
(Merck) at 10, 15 and 45C, growth in MRS broth at pH 96
or in MRS broth with 65% NaCl, hydrolysis of arginine
and by determination of the configuration of the lactic acid
enantiomer, produced using the methods of Schillinger and
Lucke (1987). Twenty-two Gram-positive, catalase-negative
cocci, which did not produce gas from glucose, grew at 45C
and in MRS broth with 65% NaCl and produced more than
90% L(+)-lactic acid, were presumptively identified as
enterococci.
Fermentation of sugars was tested using the API 20 Strep
1 system (bioMerieux Germany, Nurtingen, Germany)
according to the manufacturers instructions. Sugar fermentation patterns allowed a presumptive identification of
enterococci to species level. As one of the isolates (BFE
2451) was irrecoverably lost after phenotypic tests, genotypic experiments were performed with only 21 isolates.
2004 The Society for Applied Microbiology, Journal of Applied Microbiology, doi:10.1111/j.1365-2672.2004.02450.x
ENTEROCOCCI FROM AFRICAN HUSSUWA
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For RAPD-PCR fingerprinting, total genomic DNA from

21 enterococci isolates and various reference strains
(Table 1) were isolated according to the methods of Pitcher
et al. (1989). Two RAPD-PCR reactions were performed for
each strain, each employing a different primer. The primers
used were: primer M13 (5-GAG GGT GGC GGT TCT3) (Huey and Hall 1989) and AP4 (5-TCA CGC TGC A3) (Andrighetto et al. 2001). DNA was amplified using
methods and amplification conditions described by Andrighetto et al. (2001). PCR products were separated by
electrophoresis on a 18% (w/v) agarose gel using 1xTBE
buffer (Sambrook et al. 1989). The gels were stained in

ethidium bromide and photographed on an u.v. transilluminator. Photopositives were digitalized by scanning and
scanned images were normalized and subsequently analysed
using the Bionumerics (version 25) software package
(Applied Maths, SintMartensLatem, Belgium). Groupings of the RAPD-PCR fingerprints were performed by
means of the Pearson productmoment correlation coefficient (r) and the unweighted pair-group method using
arithmetic averages clustering algorithm (UPGMA) (Sneath
and Sokal 1973). The RAPD-PCR fingerprints obtained
with both primers were analysed together as a single data set
by calculating the average matrix from the two separate
Table 1 Strains used in this study

Strains
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RAPD-PCR fingerprinting
Relevant characteristics
Strains isolated from Hussuwa (this study)
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Enterococcus faecium strains from:

Fermentation A, stage 1: BFE 2201, BFE 2204 (LMG 22493), BFE 2205
Fermentation A, stage 2: BFE 2207, BFE 2211, BFE 2243, BFE 2253, BFE 2388
Fermentation A, stage 3: BFE 2240, BFE 2245, BFE 2256, BFE 2322
(LMG 22496), BFE 2466 (LMG 22497)
Fermentation A, stage 4: BFE 2215, BFE 2262
Fermentation A, stage 5: BFE 2214, BFE 2314
Fermentation A, stage 6: BFE 2296 (LMG 22494)
Fermentation B, stage 6: BFE 2480 (LMG 22498)
Fermentation B, stage 7: BFE 2302 (LMG 22495), BFE 2345
Fermentation B, stage 8: BFE 2451
E. faecium LMG 11423T
E. faecium FAIR-E 13 (LMG 20628), 20 (LMG 20635), 24, 41, 119 (LMG 20721),
128 (LMG 20730), 137 (LMG 20739), 198 (LMG 20760), 338 (LMG 20890),
349 (LMG 20901), 362 (LMG 20905), 366 (LMG 20909), 400 (LMG 20943)
E. faecium AM9M*
E. faecalis LMG 7937T
E. durans LMG 10746T
Listeria monocytogenes ATCC 7644, L. innocua WS 2258 and Staphylococcus aureus
DSM 6732b
E. faecalis MMH594
E. faecalis FAIR-E 177
E. faecalis FAIR-E 307 (LMG 20863)
E. faecium FAIR-E 3 (LMG 20618)
Indicator strains for testing bacteriocin activity

Esp+ control
Cyl+ control
Ace+ control
+
EfaA
fm , Asa1 control
Bacteriocin 31+ control
Enterocin A+ control
Enterocin B+ control
Enterocin P+ control
Enterocin L50 A and B+ control
Enterocin 1071 A and B+ control
Enterocin AS-48+ control
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E. faecalis BFE 1071

Reference strains used for genotyping
BFE, Federal Research Centre for Nutrition, Institute of Biotechnology and Molecular Biology, Karlsruhe, Germany; FAIR-E, research collection of
the EU-project FAIR-CT-3078 deposited in the BCCM/LMG Bacteria Collection Laboratorium voor Microbiologie, University of Gent, Gent,
Belgium; LMG, BCCM/LMG Bacteria Collection Laboratorium voor Microbiologie, University of Gent, Gent, Belgium; ATCC, American Type
Culture Collection; WS, Technical University Weihenstephan, Munich, Germany; DSM, Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Braunschweig, Germany.
*Deposited by Garrido Universidae, Santiago de Compostella.
8 Deposited by Shankar et al. (1999).
with the three different enzymes were created by averaging

the separate similarity matrices.
16S rDNA sequencing
Technological properties
The almost complete 16S rDNA nucleotide sequence was

determined for representative enterococci strains occurring
in the two genomic subgroups that were determined by
RAPD-PCR fingerprinting (see below) to confirm species
identification. The 16S rDNA gene was amplified by PCR
using primers as described by Cibik et al. (2000). DNA was
amplified in 50 ll volumes containing 100 ng template
DNA, 200 lM dNTPs, 25 pM of the respective primers,
25 U Pwo DNA polymerase (Peqlabs, Konstanz, Germany)
and 1xPwo polymerase buffer. DNA was amplified in 32
cycles (denaturation, 94C for 1 min; annealing, 51C for
1 min; extension, 68C for 2 min). The PCR product was
cleaned using Quantum-prep spin columns (Bio-rad,
Munich, Germany), according to the manufacturers
instructions and subsequently sequenced (GATC, Konstanz, Germany) using primers SP3, SP4 and SP5 as
described by Cibik et al. (2000). The 16S rDNA sequences
were compared with the 16S rDNA sequence of the E.
faecium type strain (LMG 11423T) using the pairwise
clustering comparison option of the Bionumerics sequence
types module.
Bacteriocin activity. Bacteriocin activity was detected by

the deferred inhibition assay (Ahn and Stiles 1990) with
enterococci as the producing organisms and Listeria spp. or
Staphylococcus aureus DSM 6732 (Table 1) as indicator
strains. Indicator bacteria were inoculated (1%) into soft
(075%) MRS agar, which was used to overlayer MRS agar
plates (Franz et al. 1999b). In addition, PCR amplification of
known enterocin genes was performed using total genomic
DNA from producer strains as template. The PCR primer
annealing conditions and primers used are shown in
Table 2. DNA was amplified in 50 ll volumes and the
PCR mixture contained template DNA, dNTPs and primer
concentrations as described above for the 16S rDNA
amplification. However, instead of using Pwo DNA polymerase, 15 U of Taq DNA polymerase and 1xTaq polymerase
buffer (Amersham Pharmacia, Freiburg, Germany) were
used. DNA was amplified in 35 cycles (denaturation, 94C,
1 min; annealing, at temperatures shown in Table 2, 1 min;
extension, 72C, 40 s). As controls, the bacteriocin genes
from known enterocin producers (Table 1) were amplified.
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The 16S/23S intergenic spacer region (ISR) was amplified

by PCR using primers 16S14F and 231R as described by
Zavaleta et al. (1996). Primers 16S14F and 23S1R, complementary to target sequences at ca 140 nucleotides from the
3-end of the 16S rRNA gene and ca 120 nucleotides from
the 5-end of the 23S rRNA gene, respectively, were used to
amplify the ISR of enterococci strains from Hussuwa and
other reference strains (Table 1). DNA of all enterococci
strains from Hussuwa and other reference strains (Table 1)
was amplified in 35 cycles (denaturation, 94C for 1 min;
annealing, 60C for 2 min 30 s; extension at 68C for
1 min). The reaction mixtures contained template DNA,
enzyme, buffer and dNTPs at concentrations as described
for 16S rDNA amplification above.
PCR products were digested with the restriction enzymes
Sau3AI, MseI and a-TaqI (New England Biolabs, Frankfurt
am Main, Germany) for 2 h according to manufacturers
instructions. The digested products were separated by
electrophoresis on 3% MoSieve agarose (Peqlab, Konstanz,
Germany) gels in 1x TBE buffer. The gels were analysed
with Bionumerics using the Dice correlation coefficient (sD)
and UPGMA clustering to construct the dendrogram. A
combined data set of fingerprints obtained after digestion
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RFLP analysis of the 16S/23S rDNA intergenic

spacer region
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similarity matrices for both primer fingerprint sets to obtain

a single dendrogram.
Production of hydrogen peroxide. Five microlitres of an

Enterococcus culture were spotted onto MRS agar containing
05 mmol l)1 of 2-2 azino-di-(3 ethylbenzthiazoline-6-sulphonic acid) (ABTS; Sigma, Deisenhofen, Germany) and
3 mg l)1 horseradish peroxidase (Sigma) according to the
method of Marshall (1979). The plates were incubated at
37C for 20 h. The horseradish peroxidase in the medium
oxidizes ABTS in the presence of hydrogen peroxide to
form a purple pigment in and surrounding the producing
colony.
Utilization of nondigestible a-galactoside sugars. Enterococci were grown in modified MRS medium (Gilliland
and Speck 1977), which did not contain meat extract and
glucose. Diammonium hydrogen citrate in this medium was
replaced with sodium citrate and 0004% chlorophenol red
(5 ml of a 08% ethanolic solution per litre) was added as pH
indicator. The a-galactoside sugars, stachyose or raffinose,
were added as sole carbohydrate source to this medium at a
concentration of 309 and 8 g l)1 respectively. The plates
were incubated at 37C and observed for acid production
every day over a 3-day period.
Production of a-amylase and tannase. To test for aamylase production, a single streak of a test Enterococcus
culture was made on modified MRS agar plates that did not
contain glucose, but contained 02% soluble starch instead.
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Table 2 Primer sequences, annealing temperatures and conditions for PCR amplification of enterocin genes and for adhesin to collagen from
Enterococcus faecium (acm)
Enterocin gene
Primer sequence for amplification of enterocin gene
PCR annealing temperature (C)
Product size (bp)
Enterocin A
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
56
126
Enterocin 1071 A and B

Enterocin L50 A and B
Bacteriocin 31
Enterocin AS-48
Acm
F, forward (sense) primer; R, reverse (antisense) primer.
51
52
162
120
426
98
50
123
50
340
59
1650
milk as reported previously (Franz et al. 2001). In addition,

Gel genes were amplified by PCR using primers and
amplification conditions as described by Eaton and Gasson
(2001). The production of Esp, Cyl, Efafm and adhesin of
collagen from E. faecium (Acm) was determined by PCR
amplification of the esp, cyl, Efafm and Acm genes, respectively, using primers and PCR conditions as described
previously by Franz et al. (2001) for Esp and Cyl, by Eaton
and Gasson (2001) for Efafm and by Nallapareddy et al.
(2003) for Acm. Production of AS was determined in
clumping assays as described previously (Franz et al. 2001)
or by PCR amplification of the asa1-type AS gene using
PCR primers and amplification conditions as described by
Eaton and Gasson (2001). DNAse production was tested by
streaking a loop full of culture onto Methyl Green DNAse
test agar (Difco, Heidelberg, Germany) followed by incubation of plates at 37C for 24 h. After incubation, plates
were examined for a pink zone of clearing around individual
colonies, which indicated DNAse activity. Staphylococcus
aureus DSM 6732 was used as a positive control for DNAse
testing. The positive controls used to determine production
of Esp, Ace, AS and Cyl are shown in Table 1.
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The plates were incubated at 37C overnight, after which

they were flooded with iodine. A colourless area around the
growth indicated a positive test. Bacillus subtilis strain DSM
2109 was used as a positive control and Escherichia coli
DH5a was used as a negative control. Production of tannase
was tested on brainheart infusion agar (Oxoid) containing
05% yeast extract (Oxoid) according to the method of
Osawa (1990).
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Enterocin P
50
Enterocin B
5-AAA TAT TAT GGA AAT GGA GTG TAT-3

5-GCA CTT CCC TGG AAT TGC TC-3
5-GAA AAT GAT CAC AGA ATG CCT A-3
5-GTT GCA TTT AGA GTA TAC ATT TG-3
5-TAT GGT AAT GGT GTT TAT TGT AAT-3
5-ATG TCC CAT ACC TGC CAA AC-3
5-ATA TTT AGG GGG ACC GAT AA-3
5-ATA CAT TCT TCC ACT TAT TTT T-3
5-STG GGA GCA ATC GCA AAA TTA G-3
5-ATT GCC CAT CCT TCT CCA AT-3
5-TAT TAC GGA AAT GGT TTA TAT TGT-3
5-TCT AGG AGC CCA AGG GCC-3
5-GAG GAG TIT CAT GAT TTA AAG A-3
5-CAT ATT GTT AAA TTA CCA AGC AA-3
5-GAT TTT TGA GAG ATG ATA TAG TAG-3
5-ATT CTC ATT TGT AAC GAC TAG C-3
Safety investigations
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Detection of biogenic amine production. The ability to

produce biogenic amines by decarboxylation of amino acids
was tested on a media designed by Bover-Cid and Holzapfel
(1999), which contained either of the precursor amino acids
tyrosine (free base), histidine monohydrochloride, ornithine
monohydrochloride or lysine monohydrochloride. In order
to induce decarboxylase activity before the actual screening
test, the Enterococcus strains were subcultured twice in MRS
broth containing 01% of each precursor amino acid and
0005% pyridoxal-5-phosphate. The latter was previously
shown to be important for inducing decarboxylase activity
(Recsei et al. 1985). A 5-l l)1 volume of each Enterococcus
test culture was spotted onto agar plates with and without
amino acids and plates were incubated aerobically at 37C
for 25 days. Plates were observed for a purple colour in the
producing and surrounding colonies to indicate production
of biogenic amines from precursor amino acids.
Detection of virulence factors. The production of Gel
was tested on agar plates containing either gelatin or skim
Antibiotic susceptibility testing. Antibiotic susceptibility

was tested using the E-test (Viva Diagnostika, Cologne,
Germany) according to the methods described earlier (Franz
et al. 2001). The antibiotics used included: ampicillin
(0016256 lg ml)1), benzylpenicillin (000232 lg ml)1),
chloramphenicol (0016256 lg ml)1), tetracycline (0016
256 lg ml)1), erythromycin (0016256 lg ml)1), ciprofloxacin (000232 lg ml)1), streptomycin (high range,
All E. faecium reference strains and Hussuwa isolates

clustered in a single group (group I) at a similarity level of
18%, while the E. faecalis, E. gallinarum and E. durans
reference strains clustered outside this group at 8%
similarity level (Fig. 1). Among group I strains, two
subgroups (IA and IB) could be distinguished. Strains in
subgroup IA clustered at a similarity level of 34% and
contained two of the Hussuwa E. faecium strains, i.e. BFE
2296 and BFE 2322, the reference strains E. faecium LMG
11423T (DSM 20477T) and E. faecium AM9M, as well as
strains FAIR-E 13, 41, 119, 128, 198, 137 and 338. Strains in
subgroup IB clustered at 49% similarity level and contained
19 E. faecium strains isolated from Hussuwa, as well as
strains FAIR-E 20, 24, 349, 362, 366 and 400. The
differences in the band patterns between the two subgroups
IA and IB were substantial (Fig. 1).
RESULTS
RFLP of 16S/23S ISR. Primers 16S14F and 23S1R were

used to amplify the ISR of 32 strains of E. faecium, 21 of
which were isolated from Hussuwa. The rest were wellcharacterized strains from the FAIR-E culture collection
(Vancanneyt et al. 2002) or other sources (Table 1). Groupings of RFLP patterns were performed by means of the sD
and the relationships between patterns are shown in the
UPGMA dendrogram in Fig. 2. The reproducibility of PCR
assays and running conditions estimated by analysis of
duplicate DNA extracts was 91%. The dendrogram clearly
divides the strains into four main groups (I, II, III and IV),
which were distinguished at similarity levels of 64, 70, 73
and 68% respectively. The Enterococcus strains isolated from
Hussuwa were distributed throughout all the four groups.
Group I contained strains that all clustered in group IA with
RAPD-PCR typing, while all the strains in group III and the
majority of strains in group II (with the exception of BFE
2322) clustered in group IB with RAPD-PCR typing.
Strains in group IV clustered in both group IA and IB with
RAPD-PCR typing (Figs 1 and 2).
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Phenotypic identification. A total of 22 of 210 strains

(104%), isolated during the Hussuwa fermentation, were
presumptively identified as enterococci on the basis of
phenotypic characterization. All isolates possessed the
phenotypic traits that are commonly used to identify the
enterococci, i.e. they were Gram-positive, catalase-negative
cocci, which were able to grow in the presence of 65%
NaCl, at pH 96, at both 10 and 45C and these strains
produced L(+) -lactic acid. Strains were then presumptively
identified to species level using the API 20 Strep system,
which allocated all strains to E. faecium with very good
identification.
Phenotypic and genotypic species

characterization
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00641024 lg ml)1), gentamycin (high range, 0064

1024 lg ml)1), and vancomycin (016256 lg ml)1). The
minimum inhibitory concentration (MIC) determination
was based on the reference, agar dilution method, described
in performance standards for antimicrobial susceptibility
testing [Ninth informal supplement, National Committee
for Clinical Laboratory Standards (NCCLS), Vol. 19 (10),
1999]. Resistance was interpreted according to the values
supplied in this document for enterococci, i.e. resistance to
ampicillin, penicillin and tetracycline at an MIC
16 lg ml)1; chloramphenicol and vancomycin, MIC of
32 lg ml)1; erythromycin, MIC 8 lg ml)1; ciprofloxacin, MIC 4 lg ml)1; high level gentamycin MIC
>500 lg ml)1; and high level streptomycin, MIC
>1000 lg ml)1.
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16S rDNA sequencing. The almost complete 16S rDNA

nucleotide sequences of six selected enterococci strains from
RAPD subclusters IA and IB (see below) were determined.
Using Bionumerics, these sequences were compared with
the 16S rDNA sequence of the type strain E. faecium DSM
20477T and thus the E. faecium strains BFE 2204, BFE
2466, BFE 2296, BFE 2480, BFE 2302 and BFE 2322
showed 992, 999, 999, 988, 997 and 984%, respectively,
sequence similar to that of the type strain.
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Genotypic strain characterization

RAPD-PCR fingerprinting. A total of 21 enterococci were
analysed by RAPD-PCR fingerprinting with primers M13
and AP4, and species-specific profiles for all considered
species were obtained. The reproducibility of RAPD-PCR
analyses and running conditions were estimated from
duplicate DNA extracts of the E. faecium type strain to be
95%. Figure 1 shows the UPGMA dendrogram.
Potential technological properties for application as

starter cultures. Six enterococci strains, from this study,
showed antagonistic activity against target strains in the
deferred inhibition assay (Table 3). All these six strains
possessed the enterocin L50 A and B structural genes, while
five of these strains also possessed the enterocin P gene. Two
were able to produce hydrogen peroxide (Table 3). Five of
the Enterococcus strains isolated from Hussuwa were able to
ferment both raffinose and stachyose, while one strain was
capable of fermenting stachyose only. None of these strains
produced a-amylase or tannase (Table 3).
Safety investigations. Two of the 22 isolates produced
detectable quantities of tyramine (Table 3). Production of
other biogenic amines could not be demonstrated. None of
Enterococcus durans
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100
80
60
40
20
r x 100
LMG 10746T
Enterococcus gallinarum LMG 13129T

Enterococcus faecalis
LMG 7937T
FAIR-E 128
Enterococcus faecium
FAIR-E 13
FAIR-E 41
FAIR-E 137
FAIR-E 198
PR
FAIR-E 119
DSM 20477T
LMG 11423T
IA
BFE 2296
BFE 2322
FAIR-E 338
FAIR-E 24
FAIR-E 400
TE
EC
RR
CO
UN
AM9M
FAIR-E 20
FAIR-E 362
FAIR-E 366
FAIR-E 349
BFE 2256
BFE 2214
BFE 2205
BFE 2204
BFE 2243
BFE 2240
BFE 2466
IB
BFE 2245
BFE 2215
BFE 2262
BFE 2480
BFE 2314
BFE 2302
BFE 2345
BFE 2201
BFE 2207
BFE 2211
BFE 2388
BFE 2253
Fig. 1 Dendrogram obtained by UPGMA of correlation value r of combined RAPD patterns of Enterococcus isolates from Hussuwa and reference
strains obtained with primers M13 and AP4
100
80
80
70
60
TaqI
MseI
Sau3AI
LMG 11423
BFE 2296
FAIR-E 137
FAIR-E 128
BFE 2215
BFE 2214
BFE 2288
BFE 2322
BFE 2204
BFE 2262
BFE 2466
BFE 2322
BFE 2253
BFE 2256
BFE 2201
BFE 2314
BFE 2302
BFE 2480
BFE 2243
BFE 2207
BFE 2245
BFE 2205
FAIR-E 119
FAIR-E 41
AM9M
FAIR-E 400
OO
F
II
PR
TE
E-178
IV
FAIR-E 366
FAIR-E 362
BFE 2345
FAIR-E 24
BFE 2211
EC
III
RR
Fig. 2 Dendrogram obtained by UPGMA of similarity value sD of restriction fragment length polymorphism of combined 16S/23S intergenic spacer
region patterns of Enterococcus isolates from Hussuwa and reference strains
Table 3 Technological properties of Enterococcus faecium strains isolated from Hussuwa
CO
strains (BFE)
Bacteriocin activity
against L. monocytogenes
ATCC 7644, L. innocua
H2O2
WS 2258 and S. aureus
production DSM 6732*
EntA, EntB, Ent

Amylase and
1071 A and B, AS)48,
EntL50 tannase
Raffinose Stachyose
bacteriocin 31
EntP A and B production
utilization utilization
)
)
)
)
+
+
)
+
+
+
)
)
)
)
)
)
)
)
)
+
+
+
)
)
)
)
)
)
)
)
+
+
+
)
)
)
+
+
+
)
)
+
UN
2204, 2205, 2214, 2215,

2240, 2243, 2245, 2262,
2296, 2345, 2451
2201, 2256, 2314
2466
2480
2207, 2211, 2253, 2388
2322
2302
Presence of genes for bacteriocin

production
+
+
)
)
Bacteriocin activity tested by deferred inhibition assay. Activity (+) was indicated as zone of inhibition measuring more than 1 mm from the edge of
the producer colony. Inhibitory zones ranged from 1 to 2 mm for S. aureus DSM 6732 and 36 mm for Listeria strains.
*See Table 1 for strains used in this study.
Table 4 Presence of virulence determinants and antibiotic resistance of Enterococcus faecium strains from Hussuwa
2345
2296
2207, 2243, 2245, 2262, 2322, 2451
2204, 2205
2466
2211, 2215, 2240, 2253, 2256, 2388
2314
2214
2302
2201
2480
)
)
)
)
Em
Em
Pen
Pen
Cipr, Van
Pen, Cipr, Van
Em, Cipr, Van
)
)
)
)
)
)
)
)
)
)
)
Tyramine
production
Esp
)
)
)
+
)
)
)
)
+
)
)
)
)
)
)
+
)
)
)
)
)
+
OO
F
Antibiotic
resistance
PCR amplification of virulence gene
Acm
Cyl, Asa)1, Gel
EfaAfm
+
)
+
+
+
+
+
+
+
+
+
)
)
)
)
)
)
)
)
)
)
)
)
+
+
+
+
+
)
+
+
+
+
PR
E. faecium strain (BFE)
Plate assay for

gelatinase and
DNAse production
be identified as E. faecium. Similar to our results,

Mohammed et al. (1991) showed that the microflora from
the fermented sorghum sourdough consisted of 5% enterococci and these were also identified as E. faecium. Hamad
et al. (1997) found E. faecalis (35% of isolates) as part of the
microflora of spontaneously fermented sorghum sourdough.
Thus, the enterococci appear to be often associated with
fermented sorghum-type products from Africa, although at a
low incidence compared with that of other LAB.
More than 60% of the E. faecium strains in this study
were able to ferment arabinose, while none were capable of
fermenting sorbitol. Arabinose-positive and sorbitol-negative enterococci strains have previously been described as
indicative of a human origin (Devriese and Pot 1995),
especially where enterococcal species other than E. faecium
and E. faecalis are infrequent (Devriese and Pot 1995). This
may mean that the E. faecium strains isolated from Hussuwa
could stem from human sources and that these may indicate
unsanitary conditions during preparation of the fermented
food.
RAPD-PCR was previously successfully used for the
identification of clinical and food isolates of enterococci
2 (Descheemaeker et al. 1997; Vancanneyt et al. 2002). In this
study, RAPD-PCR patterns proved to be useful also for
species identification and to reveal a considerable degree of
genomic diversity throughout the genus Enterococcus. The
presence of two intraspecies genomic groups (subgroups IA
and IB) among E. faecium strains in our study confirmed the
results obtained by Vancanneyt et al. (2002), who investigated E. faecium strains collected from foods from different
European countries and medical isolates. Some of these
CO
RR
EC
TE
the Enterococcus strains in this study produced the virulence

factors Gel or DNAse. As determined by PCR, none of the
strains possessed the genes for Cyl or AS. Two strains were
shown to possess the genes for the Esp adhesin, while all
except for one (BFE 2296) possessed the gene for the
adhesin Acm (Table 4). As for Acm, the gene for the
production of the putative virulence factor Efafm appeared to
be common among the Enterococcus strains (95%), i.e. in all
strains except strains BFE 2314 and BFE 2345, the gene for
this antigen could be detected (Table 4). Most of the
enterococci were susceptible to many of the antibiotics
tested, especially the clinically relevant antibiotics ampicillin, vancomycin and the aminoglycoside antibiotics. However, three strains (BFE 2201, 2214 and 2314) were resistant
(>32 lg ml)1) to penicillin, three (BFE 2201, 2302 and
2480) (>32 lg ml)1) to ciprofloxacin and three (BFE 2201,
2302 and 2480) to vancomycin (>256 lg ml)1). Numerous
strains (BFE 2211, 2215, 2240, 2253, 2256, 2388, 2466 and
2480) were resistant to erythromycin (>8 lg ml)1).
Esp, enterococcal surface protein; Ace, adhesin to collagen; Cyl, cytolysin; asa-1, aggregation substance type-1; Gel, gelatinase; EfaAfm, E. faecium
endocarditis antigen.
Pen, penicillin resistance (16 lg ml)1); Cipr, ciprofloxacin resistance (4 lg ml)1); Em, erythromycin resistance (8 lg ml)1); Van, vancomycin
resistance (32 lg ml)1).
DISCUSSION
UN
While the association of enterococci with European fermented foods is well known (Cogan et al. 1997; Franz et al.
1999a), little is known about the involvement of these
bacteria in African fermented foods. Enterococci were shown
in this study to form a relevant component of the microflora
of Sudanese Hussuwa, as these bacteria could be isolated
throughout the fermentation process. Based on phenotypic
characterization, RAPD-PCR fingerprinting and 16S rDNA
sequencing, the enterococci isolated from Hussuwa could all
10 N . M . K . Y O U S I F ET AL.
PR
OO
F
E. faecium strains from Hussuwa were shown to produce

H2O2.
Stachyose and raffinose are oligosaccharides, which are
resistant to cooking and other small-scale processing steps
(Holzapfel 1997). They possess a-D-galactosidic bonds that
may be hydrolysed by a-galactosidases formed by bacteria
associated with the digestive tract and with fermented foods
(Holzapfel 1997). These oligosaccharides are metabolized by
bacteria in the human intestine to carbon dioxide, hydrogen
and methane, causing flatulence, diarrhoea, indigestion and
abdominal pain (Cristofaro et al. 1974; Holzapfel 1997).
Therefore, removal of these sugars is regarded as a beneficial
trait of bacteria associated with fermentation of plant foods
containing these oligosaccharides. Five E. faecium strains, in
this study, fermented both stachyose and raffinose, while
one strain could ferment only stachyose. As sorghum
contains raffinose and stachyose at 390 and 210 mg per
100 g, respectively (Scherzer and Senser 2000), their
utilization by E. faecium strains in the Hussuwa fermentation
may contribute to lowering the amounts of these sugars in
the sorghum product and thus, lead to a nutritionally more
wholesome product.
None of the E. faecium strains in this study produced aamylase, which was not surprising, because most LAB, with
the exception of the a-amylase producing Lactobacillus
amylovorus and Lactobacillus amylophilus are known to be
nonstarch degrading. The key chemical change in the
sorghum grain, responsible for the Hussuwa fermentation, is
the action of amylase enzymes on starch, the source of which
is the malted sorghum added to the sorghum flour (Dirar
1993). Malting is the process by which the amylolytic
enzymes are activated to solubilize the polymeric reserves in
the grain. This environment, where amylolytic enzymes are
already present in great amounts, would, therefore, probably
not select for micro-organisms with the ability to produce
amylase.
Biogenic amines occur in different kinds of foods such as
fishery products, cheese, fermented sausages and other
fermented foods (ten Brink et al. 1990; Halasz et al. 1994;
Nout et al. 1994), most frequently as a result of fermentation. These compounds are formed when precursor amino
acids are decarboxylated as a result of bacterial decarboxylase enzyme activity. This usually occurs in foods that
contain proteins or free amino acids and are subject to
conditions enabling microbial or biochemical activity (ten
Brink et al. 1990). Histamine and tyramine have been the
most intensively studied biogenic amines because of their
toxicological effects resulting from their vasoactive and
psychoactive properties (Bover-Cid and Holzapfel 1999).
Three of the E. faecium strains isolated from Hussuwa
produced the biogenic amine tyramine in detectable levels,
which is not surprising, as this activity is a well-known trait
3 associated with the enterococci (Joosten and Northold 1989;
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E. faecium FAIR-E strains, which represent the two

intraspecies genomic groups, were included as reference or
control strains in our study. These could again be resolved
together with the Hussuwa strains into one of the two
intraspecies groups. Thus, strains FAIR-E 13, 41, 119, 128,
137 and 198, which belonged to the genomic group I in the
study of Vancanneyt et al. (2002), clustered into genomic
subgroup IA in our study. The E. faecium FAIR-E strains
20, 24, 362, 366 and 400, which belonged to the genomic
group II in the study of Vancanneyt et al. (2002), clustered
into genomic subgroup IB in our study. Using the same
primers and similar RAPD-PCR conditions to those of
Vancanneyt et al. (2002), we were able to resolve also the
African E. faecium strains into two intraspecies genomic
groups. As these two intraspecies genomic groups were
detected among E. faecium strains isolated from such
geographically widely distributed sources, i.e. African and
European sources, they seem to represent a stable evolutionary divergence within the E. faecium species.
Intergenic spacer regions are short stretches of DNA
located between the 16S and 23S genes in the prokaryotic
rRNA loci (Neefs et al. 1990). As these regions show DNA
sequence variability between strains of the same species, it
makes them a good marker to measure short-term phylogeny
(Zavaleta et al. 1996). Therefore, the RFLP-ISR technique
was used to investigate the diversity among E. faecium
strains isolated from Hussuwa as well as some FAIR-E
strains. Strain discrimination was only considered when
RFLP patterns were <92% similar. Accordingly, there
appeared to be no clonal relatedness between all strains
investigated, as suggested by RAPD-PCR analysis. Thus,
this second fingerprinting technique showed that these
strains were all unrelated strains of the same species.
Therefore, based on RAPD-PCR and RFLP/ISR studies,
the E. faecium strains from Hussuwa seem both phylogenetically and genomically diverse. This genomic diversity
was reflected also by the diversity of strains regarding their
phenotypic traits such as the virulence factors, antibiotic
resistance profiles and technological characteristics. The
detection of several strains with antagonistic activity against
L. innocua, L. monocytogenes, S. aureus and other indicator
strains agrees with the many data available on the ability of
enterococci to produce antimicrobial compounds, mainly
bacteriocins, which are active against various spoilage and
pathogenic bacteria (Giraffa et al. 1997; Franz et al. 1999a,
2001). For five of the six bacteriocinogenic strains in this
study, the genes for more than one bacteriocin were
detected. Multiple bacteriocin production by enterococci is
frequently reported (Franz et al. 1999a; Cintas et al. 2000;
Herranz et al. 2001; De Vuyst et al. 2003). In addition to
bacteriocins, production of hydrogen peroxide may also play
a role in antagonistic activity (Dahiya and Speck 1968;
Lindgren and Dobrogosz 1990) and, in this study, two of the
PR
OO
F
Our studies on production of the Sudanese fermented

food, Hussuwa, aim to increase the stability of the
fermentation and to improve the nutritional quality of this
product by developing a starter culture preparation, consisting of LAB isolated from this food, which show
desirable functional characteristics. The enterococci isolated
from Hussuwa entailed the first part of the investigation of
the microflora of this product, while other LAB such as
lactobacilli and pediococci are currently being investigated.
Our studies on the enterococci from Hussuwa have shown
that although the enterococci comprise only a small part of
the microflora associated with this fermentation, some
strains possess interesting functional properties including
the degradation of a-galactoside sugars and production of
bacteriocins and hydrogen peroxide. The use of such
selected strains in a starter culture preparation may improve
the nutritional quality of this product by lowering the
concentration of anti-nutritive sugars and by improving the
safety of the product. However, some of the enterococci
may pose a problem in this fermentation themselves.
Although the overall incidence of confirmed virulence
factors among the E. faecium strains in this study was low,
strains bearing virulence determinants did occur. Furthermore, most strains contained the gene for the adhesin to
collagen from E. faecium, a potential virulence determinant
which has not yet been shown to play a role in pathogenesis
but may pose a safety risk. The problem of using
enterococci in such a fermentation may be further compounded by factors such as antibiotic resistance and the
possibility of transfer of resistance genes, as well as
production of biogenic amines. No single strain possessed
the desired functional characteristics and safety factors, and
antibiotic susceptibility was considered as a successful
starter strain candidate. However, strains BFE 2207, 2211,
2253 and 2388 may be considered for inclusion in a starter
culture preparation, as they produced two different bacteriocins and did not produce detectable levels of biogenic
amines or currently recognised and confirmed virulence
traits. Strain BFE 2322 could also be considered as a
possible candidate, as this culture possessed a potentially
desirable property of hydrogen peroxide production. However, the extent and conditions for production of hydrogen
peroxide and the possible relevance for the sensory
properties would have to be ascertained first. Finally, the
functionality of the potential starter strains should first be
investigated before they are used and their beneficial effects
should far outweigh the perceived risk for use of enterococci
in food production.
UN
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EC
TE
Leisner et al. 1994; Giraffa et al. 1997; Bover-Cid and

Holzapfel 1999). The level of tyramine concentration in
production of Hussuwa was not determined and presently
no conclusions can be made as to whether tyramine levels
resulting from decarboxylase activity of enterococci or other
lactobacilli in this product would be problematic.
Most of the E. faecium strains in this study did not
produce any of the confirmed enterococcal virulence factors.
All strains lacked the Cyl and Gel virulence factors, while
two strains (BFE 2480 and BFE 2466) produced the Esp
adhesin. This low occurrence of virulence determinants
among E. faecium strains is in agreement with earlier studies
by Eaton and Gasson (2001) and Franz et al. (2001), who
showed that the incidence of virulence factors among E.
faecalis strains from foods is by far greater than for E.
faecium strains from food sources. None of the E. faecium
strains in this study produced AS. This was not surprising,
as this determinant is generally associated with pheromoneresponsive plasmids, which are mostly associated with E.
faecalis strains (Franz et al. 1999a). However, many strains
produced the Acm or the Efafm, indicating a potential risk
associated with these strains should they encounter exposed
extracellular matrix or heart tissue. Although Acm was
found to be enriched in clinical isolates of E. faecium
(Nallapareddy et al. 2003), this virulence factor as well as
Efafm, have not yet been conclusively shown to contribute to
pathogenesis in animal studies (Singh et al. 1998; Nallapareddy et al. 2003).
Enterococci are well-known to be intrinsically resistant
towards antibiotics such as cephalosporins, b-lactams, sulphonamides and low levels of clindamycin and aminoglycosides (Murray 1990; Landman and Quale 1997). With the
ability of enterococci to acquire new resistance determinants,
multiple resistant strains have emerged in the last decade,
which exhibit resistance also to tetracyclines, chloramphenicol, high levels of aminoglycosides, b-lactams and vancomycin (Murray 1990; Low et al. 1994; Landman and Quale
1997). As a result of their intrinsic resistance, it was not
surprising to find that more than 50% of the strains in this
study showed resistance to at least one antibiotic and some
even to three. Several workers have suggested that such high
levels of antibiotic resistance are related to a combination of
nonprescription antibiotic usage (Shahid et al. 1985) and the
circulation of resistant isolates in environments with limited
4 sanitation facilities (Al-Jabouri and Al-Meshhadani 1985).
However, resistance to the clinically relevant antibiotics
ampicillin, the aminoglycoside antibiotics gentamycin and
streptomycin and the glycopeptide antibiotic vancomycin
among the Hussuwa isolates was generally low. Nevertheless,
three strains (strains BFE 2201, 2392 and 2480) were resistant
(>256 lg ml)1) to vancomycin and the presence of these
vancomycin-resistant enterococci in such an African fermented food is a cause for concern.
11
ACKNOWLEDGEMENTS
N.M.K. Yousif gratefully acknowledges funding from the
German Academic Exchange Service (Deutscher Akadem-
12 N . M . K . Y O U S I F ET AL.
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Journal of Applied Microbiology 2004

Molecular characterization, technological properties and

N.M.K. Yousif1, P. Dawyndt2, H. Abriouel1, A. Wijaya1, U. Schillinger1, M. Vancanneyt2, J. Swings2,

2003/1196: received 30 December 2003, revised and accepted 9 August 2004

N.M.K. YOUSIF, P. DAWYNDT, H. ABRIOUEL, A. WIJAYA, U. SCHILLINGER, M. VANCANNEYT, J. SWINGS,

on sorghum fermentation (Dirar 1993); among these,

2004 The Society for Applied Microbiology

Journal: JAM CE: Nithya

MATERIALS AND METHODS

Samples were taken from 11 different stages during two

form of dough or as pancakes. After cooling, a further half

ENTEROCOCCI FROM AFRICAN HUSSUWA

For RAPD-PCR fingerprinting, total genomic DNA from

buffer (Sambrook et al. 1989). The gels were stained in

Table 1 Strains used in this study

Strains isolated from Hussuwa (this study)

Enterococcus faecium strains from:

Indicator strains for testing bacteriocin activity

E. faecalis BFE 1071

Reference strains used for genotyping

with the three different enzymes were created by averaging

16S rDNA sequencing

The almost complete 16S rDNA nucleotide sequence was

Bacteriocin activity. Bacteriocin activity was detected by

The 16S/23S intergenic spacer region (ISR) was amplified

RFLP analysis of the 16S/23S rDNA intergenic

similarity matrices for both primer fingerprint sets to obtain

Production of hydrogen peroxide. Five microlitres of an

ENTEROCOCCI FROM AFRICAN HUSSUWA

Primer sequence for amplification of enterocin gene

PCR annealing temperature (C)

Product size (bp)

Enterocin 1071 A and B

F, forward (sense) primer; R, reverse (antisense) primer.

milk as reported previously (Franz et al. 2001). In addition,

The plates were incubated at 37C overnight, after which

5-AAA TAT TAT GGA AAT GGA GTG TAT-3

Detection of biogenic amine production. The ability to

Antibiotic susceptibility testing. Antibiotic susceptibility

All E. faecium reference strains and Hussuwa isolates

RFLP of 16S/23S ISR. Primers 16S14F and 23S1R were

Phenotypic identification. A total of 22 of 210 strains

Phenotypic and genotypic species

00641024 lg ml)1), gentamycin (high range, 0064

16S rDNA sequencing. The almost complete 16S rDNA

Genotypic strain characterization

Potential technological properties for application as

ENTEROCOCCI FROM AFRICAN HUSSUWA

Enterococcus gallinarum LMG 13129T

Table 3 Technological properties of Enterococcus faecium strains isolated from Hussuwa

EntA, EntB, Ent

2204, 2205, 2214, 2215,

Presence of genes for bacteriocin

ENTEROCOCCI FROM AFRICAN HUSSUWA

PCR amplification of virulence gene

Cyl, Asa)1, Gel

E. faecium strain (BFE)

Plate assay for

be identified as E. faecium. Similar to our results,

the Enterococcus strains in this study produced the virulence

E. faecium strains from Hussuwa were shown to produce

E. faecium FAIR-E strains, which represent the two

ENTEROCOCCI FROM AFRICAN HUSSUWA

Our studies on production of the Sudanese fermented