Hus Suwa

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Molecular characterization, technological properties andsafety aspects of enterococci from ‘Hussuwa’, an Africanfermented 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. Franz11Federal Research Centre for Nutrition, Institute of Hygiene and Toxicology, Karlsruhe, Germany, 2 BCCM/LMG Bacteria Collection,

Laboratory of Microbiology, University of Ghent, Ghent, Belgium, and 3 Department of Biotechnology, Faculty of Agriculture, Shambat

University of Khartoum, Khartoum, Sudan

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

ABSTRACT

N . M . K . Y O U S I F , P . D A W Y N D T , H . A B R I O U E L , A . W I J A Y A , U . S C H I L L I N G E R , M . V A N C A N N E Y T , J . S W I N G S ,

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 . 2 0 0 4 .

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.

INTRODUCTION

Sorghum is a drought-tolerant plant, grown in the semi-

arid tropical regions of Africa and Asia (Dogett 1988).

Many indigenous fermented foods from Sudan are based

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 : 0Æ5 ratio. The paste is left to

ferment for 12 h, after which it is cooked lightly in the

J A M 2 4 5 0 B Dispatch: 5.10.04 Journal: JAM CE: Nithya

Journal Name Manuscript No. Author Received: No. of pages: 13 PE: Raymond

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: [email protected]).

ª 2004 The Society for Applied Microbiology

Journal of Applied Microbiology 2004 doi:10.1111/j.1365-2672.2004.02450.x

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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 24–48 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 andburied 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 sweet–sour 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 awide 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 tradi-

tional cheeses produced in Mediterranean countries from

pasteurised or raw milk (Ordonez et al. 1978; Litopoulou-

Tzanetaki 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 Schiesser1987; 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 pro-

tein (Esp), adhesin to collagen from E. faecalis (Ace) and

Enterococcus endocarditis antigen (Efafs or Efafm from E.

faecalis or E. faecium respectively), determines that entero-

cocci are not considered as ‘generally recognised as safe’

(GRAS) organisms.

This study aimed to characterize the enterococci associ-

ated with the Hussuwa production, as these bacteria

constituted a relevant proportion (ca10%) of LAB isolated

from this product during fermentation. As little is known

about the involvement of enterococci with African fer-

mented 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 infermentation and whether these bacteria should be taken

into consideration when selecting a starter culture prepar-

ation for the production of Hussuwa.

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, afterwhich, serial 10-fold dilutions were made in quarter-

strength Ringer’s 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 incu-

bated 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.

Phenotypic characterization

All Gram-positive, catalase-negative bacteria isolated from

MRS agar were considered as presumptive LAB. Presump-

tive 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 9Æ6

or in MRS broth with 6Æ5% 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 45Cand in MRS broth with 6Æ5% NaCl and produced more than

90% LL(+)-lactic acid, were presumptively identified as

enterococci.

Fermentation of sugars was tested using the API 20 Strep

system (bioMerieux Germany, Nurtingen, Germany1 )

according to the manufacturer’s instructions. Sugar fermen-

tation patterns allowed a presumptive identification of

enterococci to species level. As one of the isolates (BFE

2451) was irrecoverably lost after phenotypic tests, geno-

typic experiments were performed with only 21 isolates.

2 N . M . K . Y O U S I F ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology , doi:10.1111/j.1365-2672.2004.02450.x

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RAPD-PCR fingerprinting

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 foreach strain, each employing a different primer. The primers

used were: primer M13 (5¢-GAG GGT GGC GGT TCT-

3¢) (Huey and Hall 1989) and AP4 (5¢-TCA CGC TGC A-

3¢) (Andrighetto et al. 2001). DNA was amplified using

methods and amplification conditions described by Andri-

ghetto et al. (2001). PCR products were separated by

electrophoresis on a 1Æ8% (w/v) agarose gel using 1xTBE

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

ethidium bromide and photographed on an u.v. transillu-

minator. Photopositives were digitalized by scanning and

scanned images were normalized and subsequently analysed

using the Bionumerics (version 2Æ5) software package

(Applied Maths, Sint–Martens–Latem, Belgium). Group-ings of the RAPD-PCR fingerprints were performed by

means of the Pearson product–moment correlation coeffi-

cient (r ) and the unweighted pair-group method using

arithmetic averages clustering algorithm (UPGMAUPGMA) (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 Relevant characteristics

Enterococcus faecium strains from: Strains isolated from Hussuwa (this study)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 Reference strains used for genotyping

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 6732bIndicator strains for testing bacteriocin activity

E. faecalis MMH594 Esp+ control

E. faecalis FAIR-E 177 Cyl+ control

E. faecalis FAIR-E 307 (LMG 20863) Ace+ control

E. faecalis FAIR-E 404 (LMG 20947) EfaAþfm, Asa1+ control

E. faecium FAIR-E 3 (LMG 20618) Bacteriocin 31+ control

E. faecalis FAIR-E 119 (LMG 20721) Enterocin A+ control

Enterocin B+ control

Enterocin P+ controlEnterocin L50 A and B+ control

E. faecalis BFE 1071 Enterocin 1071 A and B+ control

E. faecalis FAIR-E 77 (LMG 20681) Enterocin AS-48+ control

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.

Deposited by Shankar et al . (1999)8 .

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similarity matrices for both primer fingerprint sets to obtain

a single dendrogram.

16S rDNA sequencing

The almost complete 16S rDNA nucleotide sequence wasdetermined 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 lMM dNTPs, 25 pMM of the respective primers,

2Æ5 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 manufacturer’s

instructions and subsequently sequenced (GATC, Kon-

stanz, 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.

RFLP analysis of the 16S/23S rDNA intergenicspacer region

The 16S/23S intergenic spacer region (ISR) was amplifiedby PCR using primers 16S14F and 231R as described by

Zavaleta et al. (1996). Primers 16S14F and 23S1R, comple-

mentary 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 describedfor 16S rDNA amplification above.

PCR products were digested with the restriction enzymes

Sau3AI, MseI and a-Taq I (New England Biolabs, Frankfurt

am Main, Germany) for 2 h according to manufacturer’s

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

with the three different enzymes were created by averaging

the separate similarity matrices.

Technological properties

Bacteriocin activity. Bacteriocin activity was detected bythe 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

(0Æ75%) 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 rDNAamplification. However, instead of using Pwo DNA polym-

erase, 1Æ5 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.

Production of hydrogen peroxide. Five microlitres of an

Enterococcus culture were spotted onto MRS agar containing

0Æ5 mmol l)1 of 2-2¢ azino-di-(3 ethylbenzthiazoline-6-sul-

phonic acid) (ABTS; Sigma, Deisenhofen, Germany) and

3 mg l)1 horseradish peroxidase (Sigma) according to themethod 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. En-

terococci 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 0Æ004% chlorophenol red

(5 ml of a 0Æ8% ethanolic solution per litre) was added as pHindicator. The a-galactoside sugars, stachyose or raffinose,

were added as sole carbohydrate source to this medium at a

concentration of 3Æ09 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 a-

amylase production, a single streak of a test Enterococcus

culture was made on modified MRS agar plates that did not

contain glucose, but contained 0Æ2% soluble starch instead.



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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 brain–heart infusion agar (Oxoid) containing

0Æ5% yeast extract (Oxoid) according to the method of

Osawa (1990).

Safety investigations

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 0Æ1% of each precursor amino acid and

0Æ005% pyridoxal-5-phosphate. The latter was previouslyshown 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 2–5 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

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, respect-

ively, 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 incu-

bation 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 productionof Esp, Ace, AS and Cyl are shown in Table 1.

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

(0Æ016–256 lg ml)1), benzylpenicillin (0Æ002–32 lg ml)1),

chloramphenicol (0Æ016–256 lg ml)1), tetracycline (0Æ016–

256 lg ml)1), erythromycin (0Æ016–256 lg ml)1), cipro-

floxacin (0Æ002–32 lg ml)1), streptomycin (high range,

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: 5¢-AAA TAT TAT GGA AAT GGA GTG TAT-3¢ 56 126

R: 5¢-GCA CTT CCC TGG AAT TGC TC-3¢Enterocin B F: 5¢-GAA AAT GAT CAC AGA ATG CCT A-3¢ 50 162

R: 5¢-GTT GCA TTT AGA GTA TAC ATT TG-3¢

Enterocin P F: 5¢-TAT GGT AAT GGT GTT TAT TGT AAT-3 ¢ 50 120

R: 5¢-ATG TCC CAT ACC TGC CAA AC-3¢

Enterocin 1071 A and B F: 5¢-ATA TTT AGG GGG ACC GAT AA-3¢ 51 426

R: 5¢-ATA CAT TCT TCC ACT TAT TTT T-3¢

Enterocin L50 A and B F: 5¢-STG GGA GCA ATC GCA AAA TTA G-3 ¢ 52 98

R: 5¢-ATT GCC CAT CCT TCT CCA AT-3¢

Bacteriocin 31 F: 5¢-TAT TAC GGA AAT GGT TTA TAT TGT-3 ¢ 50 123

R: 5¢-TCT AGG AGC CCA AGG GCC-3¢

Enterocin AS-48 F: 5¢-GAG GAG TIT CAT GAT TTA AAG A-3¢ 50 340

R: 5¢-CAT ATT GTT AAA TTA CCA AGC AA-3¢

Acm F: 5¢-GAT TTT TGA GAG ATG ATA TAG TAG-3¢ 59 1650

R: 5¢-ATT CTC ATT TGT AAC GAC TAG C-3¢

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



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0Æ064–1024 lg ml)1), gentamycin (high range, 0Æ064–

1024 lg ml)1), and vancomycin (0Æ16–256 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 Committeefor 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; ciprofloxa-

cin, MIC ‡4 lg ml)1; high level gentamycin MIC

>500 lg ml)1; and high level streptomycin, MIC

>1000 lg ml)1.

RESULTSPhenotypic and genotypic speciescharacterization

Phenotypic identification. A total of 22 of 210 strains

(10Æ4%), 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 6Æ5%

NaCl, at pH 9Æ6, at both 10 and 45C and these strains

produced LL(+) -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.

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 99Æ2, 99Æ9, 99Æ9, 98Æ8, 99Æ7 and 98Æ4%, respectively,

sequence similar to that of the type strain.

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 UPGMAUPGMA dendrogram.

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 insubgroup 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).

RFLP of 16S/23S ISR. Primers 16S14F and 23S1R wereused to amplify the ISR of 32 strains of E. faecium, 21 of

which were isolated from Hussuwa. The rest were well-

characterized strains from the FAIR-E culture collection

(Vancanneyt et al. 2002) or other sources (Table 1). Group-

ings of RFLP patterns were performed by means of the sDand 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).

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, whilefive 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



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1 0 0

8 0

6 0

4 0

2 0

Enterococcus faecium







Enterococcus durans

Enterococcus gallinarum

Enterococcus faecalis










LMG 10746T

LMG 13129T

LMG 7937T

FAIR-E 128

FAIR-E 13

FAIR-E 41

FAIR-E 137

FAIR-E 198

FAIR-E 119

DSM 20477T

LMG 11423T

BFE 2296

AM9M

FAIR-E 338

FAIR-E 24

FAIR-E 400

FAIR-E 20

FAIR-E 362

FAIR-E 366

FAIR-E 349

BFE 2256

BFE 2214

BFE 2205

BFE 2204

BFE 2243

BFE 2466

BFE 2245

BFE 2215

BFE 2262

BFE 2480

BFE 2302

BFE 2345

BFE 2201

BFE 2207

BFE 2211

BFE 2388

BFE 2253

BFE 2322

BFE 2240

BFE 2314

IA

IB

r x 100

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



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Taq I Mse I Sau3AI

Enterococcus faecium LMG 11423

BFE 2296

BFE 2215BFE 2214

BFE 2288

BFE 2322

BFE 2204

BFE 2466

BFE 2322

BFE 2253

BFE 2256

BFE 2314

BFE 2302

BFE 2480

BFE 2243

BFE 2207

BFE 2245BFE 2205

BFE 2262

FAIR-E 137

FAIR-E 119

BFE 2201

FAIR-E 41

FAIR-E 400

FAIR-E 366

FAIR-E 24

BFE 2211

FAIR-E 362

BFE 2345

AM9M

FAIR-E 128

Enterococcus faecium Enterococcus faecium

Enterococcus faecium Enterococcus faecium Enterococcus faecium

Enterococcus faecium Enterococcus faecium Enterococcus faecium



E-178

6 0

7 0

8 0

8 0

1 0 0

I

II

III

IV

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


strains (BFE)

H2O2

production

Bacteriocin activity

against L. monocytogenes

ATCC 7644, L. innocua

WS 2258 and S. aureus

DSM 6732*

Presence of genes for bacteriocin

production

Amylase and

tannase

production

Raffinose

utilization

Stachyose

utilization

EntA, EntB, Ent

1071 A and B, AS)48,

bacteriocin 31 EntP

EntL50

A and B

2204, 2205, 2214, 2215,

2240, 2243, 2245, 2262,2296, 2345, 2451

) ) ) ) ) ) ) )

2201, 2256, 2314 ) ) ) ) ) ) + +

2466 ) + ) + ) + +

2480 ) + ) + + ) + +

2207, 2211, 2253, 2388 ) + ) + + ) ) )

2322 + ) ) ) ) ) ) )

2302 + ) ) ) ) ) ) +

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 3–6 mm for Listeria strains.

*See Table 1 for strains used in this study.



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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 forthis antigen could be detected (Table 4). Most of the

enterococci were susceptible to many of the antibiotics

tested, especially the clinically relevant antibiotics ampicil-

lin, vancomycin and the aminoglycoside antibiotics. How-

ever, 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).

DISCUSSION

While the association of enterococci with European fer-

mented 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

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% entero-

cocci 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-negat-

ive 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 theidentification of clinical and food isolates of enterococci

(Descheemaeker et al. 19972 ; 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 investi-

gated E. faecium strains collected from foods from different

European countries and medical isolates. Some of these

Table 4 Presence of virulence determinants and antibiotic resistance of Enterococcus faecium strains from Hussuwa

E. faecium strain (BFE)

Antibiotic

resistance

Plate assay for

gelatinase and

DNAse production

Tyramine

production

PCR amplification of virulence gene

Esp Acm Cyl, Asa)1, Gel EfaAfm

2345 ) ) ) ) + ) )

2296 ) ) ) ) ) ) +

2207, 2243, 2245, 2262, 2322, 2451 ) ) ) ) + ) +

2204, 2205 ) ) + ) + ) +

2466 Em ) ) + + ) +

2211, 2215, 2240, 2253, 2256, 2388 Em ) ) ) + ) +

2314 Pen ) ) ) + ) )

2214 Pen ) ) ) + ) +

2302 Cipr, Van ) + ) + ) +

2201 Pen, Cipr, Van ) ) ) + ) +

2480 Em, Cipr, Van ) ) + + ) +

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).



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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 thestudy 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 evolu-tionary 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 phyloge-

netically 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, mainlybacteriocins, 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

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-DD-galactosidic bonds that

may be hydrolysed by a-galactosidases formed by bacteriaassociated 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), theirutilization 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 a-

amylase, 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 amylolyticenzymes 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 fermenta-

tion. These compounds are formed when precursor amino

acids are decarboxylated as a result of bacterial decarboxy-

lase enzyme activity. This usually occurs in foods thatcontain 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

associated with the enterococci (Joosten and Northold 19893 ;

10 N . M . K . Y O U S I F ET AL.


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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 pheromone-responsive 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; Nallapa-

reddy et al. 2003).

Enterococci are well-known to be intrinsically resistant

towards antibiotics such as cephalosporins, b-lactams, sul-

phonamides and low levels of clindamycin and aminoglyco-sides (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, chloramphen-

icol, high levels of aminoglycosides, b-lactams and vancomy-

cin (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 thecirculation of resistant isolates in environments with limited

sanitation facilities (Al-Jabouri and Al-Meshhadani 19854 ).

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 fer-

mented food is a cause for concern.

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, con-

sisting of LAB isolated from this food, which show

desirable functional characteristics. The enterococci isolatedfrom 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 thesafety 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. Further-

more, 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 com-

pounded by factors such as antibiotic resistance and the

possibility of transfer of resistance genes, as well as

production of biogenic amines. No single strain possessedthe 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 bacte-

riocins 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. How-

ever, the extent and conditions for production of hydrogen

peroxide and the possible relevance for the sensoryproperties 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.

ACKNOWLEDGEMENTS

N.M.K. Yousif gratefully acknowledges funding from the

German Academic Exchange Service (Deutscher Akadem-



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ischer Austausch Dienst – DAAD) under the Sandwich

Programme.

REFERENCES

Ahn, C. and Stiles, M.E. (1990) Plasmid-associated bacteriocinproduction by a strain of Carnobacterium piscicola from meat. Applied

and Environmental Microbiology 56, 2503–2510.

Al-Jabouri, M.M. and Al-Meshhadani, N.S. (1985) A note on

antibiotic-resistant Echerichia coli in adult man, raw sewage and

sewage polluted River Tigris in Mosul (Iraq). Journal of Applied

bacteriology 59 , 513–518.

Andrighetto, C., Knijff, E., Lombardi, A., Torriani, S., Vancanneyt,

M., Kersters, K., Swings, J. and Dellaglio, F. (2001) Phenotypic and

genetic diversity of enterococci isolated from Italien cheeses. Journal

of Dairy Research 68, 303–316.

Bover-Cid, S. and Holzapfel, W.H. (1999) Improved screening

procedure for biogenic amine production by lactic acid bacteria.

International Journal of Food Microbiology 53, 33–41.

ten Brink, B. Damink, C., Joosten, H.M.L.J. and Huis in’t veld, J.H.J.

(1990) Occurence and formation of biologically active amines in

foods. International Journal of Food Microbiology 11, 73–84.

Centeno, J.A., Menendez, S. and Rodrıguez-Otero, J.L. (1996) Main

microbial flora present as natural starters in cebreiro raw cow’s milk

cheese (northwest Spain). International Journal of Food Microbiology

33, 307–313.

Cibik, R., Lepage, E. and Tailliez, P. (2000) Molecular diversity of

Leuconostoc mesenteroides and Leuconostoc citreum isolated from

traditional French cheeses as revealed by RAPD fingerprinting,

16S rDNA sequencing and 16S rDNA fragment amplification.

Systematic and Applied Microbiology 23, 267–278.

Cintas, L.M., Casaus, P., Herranz, C., Havarstein, L.S., Holo, H.,

Hernandez, P. and Nes, I.F. (2000) Biochemical and geneticevidence that Enterococcus faecium L50 produces enterocins L50A

and L50B, the sec-dependent enterocin P, and a novel bacteriocin

secreted without an N-terminal extension termed enterocin Q.

Journal of Bacteriology 182, 6806–6814.

Cogan, T.M., Barbosa, M., Beuvier, E., Bianchi-Salvadori, B.,

Cocconcelli, P.S., Fernandez, I., Gomez, J., Gomez, R. et al.

(1997) Characterization of the lactic acid bacteria in artisanal dairy

products. Journal of Dairy Research 64, 409–421.

Cristofaro, E., Mottu, F. and Wuhrmann, J.J. (1974) ???. In Sugars in

Nutrition ed. Sipple, H.L. and McNutt, K. p. 313. New York:

Academic Press.5

Dahiya, R.S. and Speck, M.L. (1968) Hydrogen peroxide formation by

lactobacilli and its effect on Staphylococcus aureus. Journal of Dairy

Science 51, 1568–1572.

De Vuyst, L., Foulquie Moreno, M.R. and Revets, H. (2003)

Screening for enterocins and detection of hemolysin and vancomycin

resistance in enterococci of different origins. International Journal of

Food Microbiology 84 , 299–318.

Descheemaeker, P., Lammens, C., Pot, B. Vandamme, P. and

Goossens, H. (1997) Evaluation of arbitrarily primed PCR analysis

and pulsed-field gel electrophoresis of large genomic DNA

fragments for identification of enterococci important in human

medicine. International Journal of Systematic Bacteriology 47, 555–

561.

Devriese, L.A. and Pot, B. (1995) The genus Enterococcus. In The

Genera of Lactic Acid Bacteria, Vol. 2. ed. Wood, B.J.B. and

Holzapfel, W.H. pp. 327–367. London: Blackie Academic.

Dirar, H.A. (1993) The Indigenous Fermented Foods of the Sudan.

Wallingford, UK: CAB International.

Dogett, H. (1988) Sorghum, pp 5–10. New York: John Wiley & Sons

Inc.

Eaton, T.J. and Gasson, M.J. (2001) Molecular screening of

Enterococcus virulence determinants and potential for genetic

exchange between food and medical isolates. Applied and Environ-

mental Microbiology 67 , 1628–1635.

Franz, C.M.A.P., Holzapfel, W.H. and Stiles, M.E. (1999a) Entero-

cocci at the crossroads of food safety? International Journal of Food

Microbiology 47, 1–24.

Franz, C.M.A.P., Worobo, R.W., Quadri, L.E.N., Schillinger, U.,

Holzapfel, W.H., Vederas, J.C. and Stiles, M.E. (1999b) Atypical

genetic locus for enterocin B production in Enterococcus faecium BFE

900. Applied and Environmental Microbiology 65, 2170–2178.

Franz, C.M.A.P., Muscholl-Silberhorn, A.B., Yousif, N.M.K.,

Vancanneyt, M., Swings, J. and Holzapfel, W.H. (2001) Incidenceof virulence factors and antibiotic resistance among enterococci

isolated from food. Applied and Environmental Microbiology 67,

4385–4389.

Gilliland, S.E. and Speck, M.L. (1977) Use of Minitek-system for

characterizing lactobacilli. Applied and Environmental Microbiology

33, 1289–1292.

Giraffa, G., Carminati, D. and Neviani, E. (1997) Enterococci isolated

from dairy products: a review of risks and potential technological

use. Journal of Food Protection 60, 732–738.

Halasz, A., Barath, A., Simon-Sarkadi, L. and Holzapfel, W.H. (1994)

Biogenic amines and their production by microorganisms in food.

Trends in Food Science and Technology 5, 42–49.

Hamad, S.H., Dieng, M.C., Ehrmann, M.A. and Vogel, R.F. (1997)

Characterisation of the bacterial flora of Sudanese sorghum flour and

sorghum sourdough. Journal of Applied Microbiology 83 , 764–770.

Herranz, C., Mukhopadhyay, S., Casaus, P., Martınez, J.M., Rodrı-

guez, J.M., Nes, I.F., Hernandez, P.E. and Cintas, L.M. (2001)

Enterococcus faecium P 21: a strain occurring naturally in dry-

fermented sausages producing the class II bacteriocins enterocin A

and enterocin B. Food Microbiology 18, 115–131.

Holzapfel, W.H. (1997) Use of starter cultures in fermentation on a

household scale. Food Control 8, 241–258.

Huey, B and Hall, J. (1989) Hypervariable DNA fingerprinting in

Escherichia coli . Minisatellite probe from bacteriophage M13. Journal

of Bacteriology 17, 2528–2532.

Joosten, H.M.L.J. and Northold, M.D. (1989) Detection, growth and

amine-producing capacity of lactobacilli in cheese. Applied and Environmental Microbiology 55, 2356–2362.

Landman, D. and Quale, J.M. (1997) Management of infections due to

resistant enterococci: a review of therapeutic options. Journal of

Antimicrobial Agents and Chemotherapy 40, 161–170.

Leclercq, R. (1997) Enterococci acquire new kinds of resistance.

Clinical Infectious Diseases 24 , S80–S84.

Leisner, J.J., Millan, J.C. Huss, H.H. and Larsen, L.M. (1994)

Production of histamine and tyramine by lactic acid bacteria isolated

from vacuum-packaged sugar-salted fish. Journal of Applied Bac-

teriology 76 , 417–423.

12 N . M . K . Y O U S I F ET AL.


8/17/2019 Hus Suwa


U

N

O

R

R

E

T

E

P

R

O

O

F

Lindgren, S.E. and Dobrogosz, W.J. (1990) Antagonistic activities of

lactic acid bacteria in food and feed fermentations. FEMS Micro-

biology Reviews 87 , 149–164.

Litopoulou-Tzanetaki, E. (1990) Changes in numbers and kinds of

lactic acid bacteria during ripening of Kefalotyri cheese. Journal of

Food Science 55, 111–113.

Low, D.E., Willey, B.M., Betschel, S. and Kreiswirth, B. (1994)

Enterococci: pathogens of the 90s. European Journal of Surgery ??

(Suppl. 573), 19–24.6

Marshall, V.M. (1979) A note on screening hydrogen peroxide-

producing lactic acid bacteria using a non-toxic chromogen. Journal

of Applied Bacteriology 47, 327–328.

Mohammed, S.I., Steenson, L.R. and Kirleis, A.W (1991) Isolation

and characterization of microorganisms associated with the tradi-

tional sorghum fermentation for production of Sudanese Kisra.

Applied and Environmental Microbiology 57 , 2529–2533.

Morrison, D., Woodford, N. and Cookson, B. (1997) Enterococci as

emerging pathogens of humans. Journal of Applied Microbiology

Symposium Supplement 83, 89S–99S.

Murray, B.E. (1990) The life and times of Enterococcus. Clinical Microbiology Reviews 3, 46–65.

Nallapareddy, S., Weinstock, G.M. and Murray, B.E. (2003) Clinical

isolates of Enterococcus faecium exhibit strain-specific collagen

binding mediated by Acm, a new member of the MSCRAMM

family. Molecular Microbiology 47 , 1733–1747.

Neefs, J.M., Van de Peer, Y., Hendriks, L. and De Wachter, R. (1990)

Compilation of small ribosomal RNA sequences. Nucleic Acids

Research 18 , 2237–2318.

Nout, M.J.R., Nche, P.F. and Hollman, P.C.H. (1994) Investigation of

the presence of biogenic amines and ethyl carbamate in Kenkey

made from maize and maize-cowpea mixtutes as influenced by

process conditions. Food Additives and Contaminants 11, 397–402.

Onyekwere, O., Akinrele, I.A. and Koleoso, O.A. (1989) Industrial-

ization of indigenous knowledge. In Food Processing Technologies for

Africa, pp. 103–120. ???: UNIDO.7

Ordonez, J.A., Barneto, R. and Ramos, M. (1978) Studies on

Manchego cheese ripened in olive oil. Milchwissenschaft 33, 609–612.

Osawa, R. (1990) Formation of a clear zone on tannin-treated brain

heart infusion agar by a Streptococcus sp. isolated from feces of

Koalas. Applied and Environmental Microbiology 56, 829–831.

Pitcher, D.G., Saunters, N.A. and Owen, R.J. (1989) Rapid extraction

of bacterial genomic DNA with guanidium thiocyanate. Letters in

Applied Microbiology 8 , 151–156.

Recsei, P.A., Moore, W.M., Snell, E.E. (1985) Pyruvol-dependent

histidine decarboxylases from Clostridium perfringens and Lactobacil-

lus buchneri . Journal of Biological Chemistry 258, 439–444.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning:

A Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold

Spring Harbor Laboratory Press.

Scherzer, H. and Senser, F. (2000) Food Composition and Nutrition

Tables, p. 565. Stuttgart: Medpharm.

Schillinger, U. and Lucke, F.-K. (1987) Identification of lactobacilli

from meat and meat products. Food Microbiology 4, 199–208.

Shahid, N.S., Rahman, M.M, Haider, K., Banu, H. and Rahman, N.

(1985) Changing pattern of resistant Shiga bacillus in Bangladesh.

Journal of Infectious Diseases 152 , 1114–1119.

Singh, K.V., Qin, X., Weinstock, G.M. and Murray, B.E. (1998)Generation and testing of mutants of Enterococcus faecalis in a

mouse peritonitis model. Journal of Infectious Diseases 178, 1416–

1420.

Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy: the

Principles and Practise of Numerical Classification. San Francisco, CA:

Freeman.

Trovatelli, L.D. and Schiesser, A. (1987) Identification and significance

of enterococci in hard cheese made from raw cow and sheep milk.

Milschwissenscaft 42, 717–719.

Vancanneyt, M., Lombardi, A., Andrighetto, C., Knijff, E., Torriani,

S., Bjorkroth, J.K., Franz, C.M.A.P., Folquie Moreno, M.R. et al.

(2002) Intra-species genomic groups in Enterococcus faecium and their

correlation with origin and pathogenicity. Applied and Environmental

Microbiology 68, 1381–1391.

Zavaleta, A.I., Martinez-Murcia, A.J. and Rodriguez-Valera, F. (1996)

16S-23S rDNA intergenic sequences indicate that Leuconostoc oenos

is phylogenetically homogenous. Microbiology 142 , 2105–2114.



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