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International Journal of Food Microbiology 88 (2003) 311–314

Short communication

Species distribution and antibiotic resistance patterns of enterococci

isolated from food of animal origin in Germany

Jan Peters*, Kiem Mac, Heidi Wichmann-Schauer, Gunter Klein, Luppo Ellerbroek

Federal Institute for Protection of Consumers and Veterinary Medicine, Diedersdorfer Weg 1, D-12277 Berlin Marienfelde, Germany

Accepted 26 February 2003

Abstract

Presently, enterococci take the third place of bacterial pathogens associated with nosocomial infections, after staphylococci

and Escherichia coli. Especially, the resistances of enterococci to several available antibiotics are threatening. We attempted to

determine which species of enterococci could be found in food of animal origin and their significance according to their

antibiotic resistances for human beings. From November 2000 to May 2002 we investigated 155 samples of food of animal

origin bought in retail outlets in Germany: 27 samples of sausages, 19 of ham, 83 of minced meat, 26 of cheese. From these

food samples we isolated 416 enterococcal strains. The most frequent species was Enterococcus faecalis (299 strains);

furthermore, we found Enterococcus faecium (54 strains), Enterococcus durans together with Enterococcus hirae (24 strains),

Enterococcus casseliflavus (22 strains), Enterococcus avium (9 strains) and Enterococcus gallinarum (8 strains). We focused on

the resistance patterns of 118 selected E. faecium and E. faecalis strains to 13 antimicrobial active agents (ampicillin,

amoxicillin/clavulanic acid, avilamycin, chloramphenicol, enrofloxacin, erythromycin, flavomycin, gentamicin, penicillin,

quinupristin/dalfopristin, teicoplanin, tetracycline and vancomycin). From the clinical point of view, the situation of antibiotic

resistance to the examined antimicrobial agents seemed to be favourable.

The investigated strains were sensitive to ampicillin and amoxicillin/clavulanic acid. These antibiotics are, in combination

with an aminoglycoside, for example gentamicin, agents of choice for the treatment of enterococcal infections in human

medicine. Only one E. faecium strain was resistant to penicillin, while all strains were sensitive to the glycopeptide antibiotics,

vancomycin and teicoplanin.

Resistances found against the antibiotics, tetracycline, quinupristin/dalfopristin and erythromycin, are causes for concern.

D 2003 Published by Elsevier B.V.

Keywords: Enterococci; GRE; Antibiotic resistance

1. Introduction

Enterococci are Gram-positive bacteria and are gen-

erally considered as low pathogenic. For immunocom-

0168-1605/$ - see front matter D 2003 Published by Elsevier B.V.

doi:10.1016/S0168-1605(03)00193-4

* Corresponding author. Tel.: +49-1888-412-2125.

E-mail address: j.peters@bfr.bund.de (J. Peters).

promised individuals, they may lead to severe diseases

like bacteremias including endocarditis, wound and

urinary tract infections. A range of natural and

acquired antibiotic resistances is characteristic for

enterococci. The natural resistance includes cepha-

losporines, aminoglycosides (low-level type), poly-

myxins, lincomycin and clindamycin (mostly). Addi-

tionally, enterococci often possess resistance or

J. Peters et al. / International Journal of Food Microbiology 88 (2003) 311–314312

intermediate susceptibility to quinolones. Furthermore,

they are able to acquire resistances to macrolides,

tetracyclines, chloramphenicol, trimethoprim/sulfame-

thoxazole, rifampicin, aminoglycosides (high-level

type) and ampicillin (Klare and Reissbrodt, 1998). At

the end of the 1980s, the first glycopeptide-resistant

enterococci (GRE) were detected, infections caused by

these strains could hardly be treated.

In addition to selection in hospitals, another poten-

tial source of acquired resistance genes is the therapeu-

tic use of antimicrobial agents in veterinary medicine or

the application as growth promoters in conventional

animal fattening (Aarestrup, 2000). These resistance

genes may effect human beings via the food chain

(Klare et al., 1995; Chadwick et al., 1996; Lemcke and

Bulte, 2000).

The objective of this study was to determine the

species distribution and the antibiotic resistance pat-

terns of enterococci isolated from food of animal origin

in Germany.1

2. Materials and methods

Between November 2000 and May 2002, 155 food

samples of animal origin were collected from different

regions of Germany. These samples included 25 sam-

ples of raw sausages, 2 of cooked sausages, 19 samples

of ham, 83 of mincedmeat and 26 of cheese. They were

bought in retail outlets, stored at + 4 jC and transferred

to the laboratory. The food sample (10 g) was homo-

genised in 90ml 1% buffered peptonewater or a special

vancomycin-resistant enterococci (VRE) enrichment

broth (CM0984 and SR0184, Oxoid,Wesel, Germany).

The homogenate (0.1 ml) was transferred to Citrate

Azide Tween Carbonate (CATC) agar (enterococci

selective; Merck, Darmstadt, Germany) and to CATC

agar with 6 mg/l vancomycin (selective for glycopep-

tide-resistant enterococci, GRE; vancomycin from

Sigma, Deisenhofen, Germany).

The agar and the remaining homogenate were incu-

bated for 24 h at 37 jC and both plates then subse-

quently incubated for a further 24 h under daylight at

room temperature.

1 This study is part of a doctoral thesis, which has not been

completed yet.

For each food sample, a maximum of three morpho-

logically different colonies of CATC and CATC+ van-

comycin were inoculated on Columbia sheep’s blood

agar (Merck).

The genus Enterococcus was confirmed using the

catalase test, pyrrolidonyl arylamidase test (Oxoid,

Basingstoke, England) and testing for growth in

BHI broth with 6.5% NaCl.

The species was determined by examining haemol-

ysis behaviour, pigment production, motility, the abil-

ity to reduce tetrazolium, the presence of arginine

dihydrolase and a series of hydrocarbon utilisations

(a-D-methyl-glucopyranoside, ribose, arabinose, raffi-

nose, mannitol and melibiose).

The Minimal Inhibitory Concentration (MIC) val-

ues were determined using the microbroth dilution

method with the Sensititre system (MCS Diagnostics,

East Grindstead, England). Because of their threat-

ening meaning in human medicine (e.g. cause of

nosocomial infections), we focused on the resistance

patterns of the two species, Enterococcus faecium and

Enterococcus faecalis. Therefore, we selected 17 E.

faecium and 101 E. faecalis strains and investigated

their sensitivity to 13 antimicrobial active agents

(ampicillin, amoxicillin/clavulanic acid, avilamycin,

chloramphenicol, enrofloxacin, erythromycin, flavo-

mycin, gentamicin, penicillin, quinupristin/dalfopris-

tin, teicoplanin, tetracycline and vancomycin).

3. Results

3.1. Species distribution

From 155 food samples we isolated 416 strains of

enterococci, which belong to the following species:

299 E. faecalis strains (72%), 54 E. faecium strains

(13%), 24 Enterococcus durans and Enterococcus

hirae strains (6%), 22 Enterococcus casseliflavus

strains (5%), 9 Enterococcus avium strains (2%) and

8 Enterococcus gallinarum strains (2%).

3.2. Antibiotic resistance patterns

The results of our studies are shown in Table 1.

All investigated strains were sensitive to ampicil-

lin, amoxicillin/clavulanic acid, vancomycin and tei-

coplanin.

Table 1

Antibiotic resistance behaviour of 118 E. faecalis and E. faecium strains isolated from 27 samples of sausages, 19 of ham, 83 of minced meat

and 26 of cheese

Antimicrobial active agent E. faecalis (n= 101) E. faecium (n= 17)

Sensitive

(%)

Intermediary

(%)

Resistant

(%)

Sensitive

(%)

Intermediary

(%)

Resistant

(%)

Ampicillin (b) 100 a 0 100 a 0

Amoxicillin/clavulanic acid (b) 100 a 0 100 a 0

Avilamycin (a) 96 a 4 71 a 29

Chloramphenicol (b) 57 36 7 71 29 0

Enrofloxacin (c) 90 9 1 12 23 65

Erythromycin (b) 26 67 7 0 82 18

Flavomycin (a) 94 a 6 b b b

Gentamicin (high-level resistance) (d) a a 0.8 a a 0

Penicillin (b) 100 a 0 94 a 6

Quinupristin/Dalfopristin (b) b b b 59 12 29

Teicoplanin (b) 100 0 0 100 0 0

Tetracycline (b) 61 1 38 82 0 18

Vancomycin (b) 100 0 0 100 0 0

Breakpoints: (a) DANMAP (2000) (b) NCCLS (1999) (c) like Ciprofloxacin, NCCLS (2001) (d) MIC>1024 Ag/ml.a No breakpoints defined.b Not tested because of the intrinsic resistance of the species against this substance.

J. Peters et al. / International Journal of Food Microbiology 88 (2003) 311–314 313

Only one E. faecalis strain among the 118 exam-

ined strains showed high-level resistance to gentami-

cin. It was isolated from ham.

All E. faecalis strains and 94% of the E. faecium

strains were sensitive to penicillin, while 6% of the E.

faecium strains was resistant to this antibiotic.

Avilamycin and flavomycin are growth promoters,

which are still used in Europe. We found comparable

sensitivity rates among the E. faecalis strains (96%

avilamycin-sensitive, 94% flavomycin-sensitive). The

E. faecium strains possess a natural resistance to fla-

vomycin (Butaye et al., 2000) and accordingly, 71%

was determined as sensitive to avilamycin. Thirty-eight

percent of the E. faecalis strains and 18% of the E.

faecium strains were resistant to tetracycline.

Quinupristin/Dalfopristin is a new antimicrobial

agent in the human medicine for the treatment of

infections with vancomycin-resistant E. faecium. E.

faecalis shows a natural resistance to this agent. Fifty-

nine percent of the E. faecium strains was sensitive,

12% intermediary and 29% resistant to quinupristin/

dalfopristin.

The use of chloramphenicol in animal husbandry is

banned in Europe. Despite this fact, we found 43%

intermediary and resistant E. faecalis strains and 29%

intermediary resistant E. faecium strains.

Chinolone antibiotics (e.g. enrofloxacin) possess a

weak effect on enterococci. We found different re-

sistance behaviours of E. faecalis and E. faecium.

While 90% of the E. faecalis strains was enroflox-

acin-sensitive, 65% of the E. faecium strains was

resistant.

Resistance to erythromycin as a representative of

the macrolide antibiotics is a matter of concern. A

high percentage of E. faecalis strains (74%) and all E.

faecium strains were intermediary or resistant.

4. Discussion

Penicillins (e.g. ampicillin and amoxicillin) are the

agents of choice in combination with aminoglycoside

(for instance gentamicin) for the treatment of enter-

ococcal infections in human medicine. The glyco-

peptide antibiotics, vancomycin and teicoplanin, are

important reserve antibiotics in case of resistance to

penicillins, high-level resistance to aminoglycosides or

allergy to penicillins.

A very ‘‘advantageous’’ result of our study is a total

sensitivity to the following clinical important antibi-

otics: ampicillin, amoxicillin/clavulanic acid, vanco-

mycin and teicoplanin.

J. Peters et al. / International Journal of Food Microbiology 88 (2003) 311–314314

Enterococci possess a natural low-level resistance

to aminoglycosides. The high-level resistance rarely

occurred. Resistance (0.8%) to gentamicin is consid-

ered as very low. The reason for the high level of

resistance to tetracycline determined in this study may

have resulted from the fact that this antibiotic is often

used as a therapeutic antimicrobial in veterinary med-

icine in Germany.

The high level resistance of E. faecium to quinu-

pristin/dalfopristin might be caused by the use of

virginiamycin in conventional animal fattening (Wer-

ner et al., 1998; Johnson et al., 2000). Virginiamycin, a

streptogramin antibiotic like quinupristin/dalfopristin,

had been used as a growth promoter. It causes cross-

resistance to quinupristin/dalfopristin (Simjee et al.,

2001; Woodford et al., 2001), and because of this fact,

it has been banned in the European Union since 1999.

Acknowledgements

We thank C. Dittmar-Gabor and C. Fester for

skillful technical assistance.

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Livermore, D.M., 2000. Quinupristin/dalfopristin and linezolid:

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ther. 46, 347–350.

Martinez, J.L., Baquero, F., 2000. Mutation frequencies and

antibiotic resistance. Antimicrob. Agents Chemother. 44,

1771–1777.