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Applied and Environmental Microbiology, October 2005, p. 6407-6409, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6407-6409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

High Prevalence of VanA-Type Vancomycin-Resistant Enterococci in Austrian Poultry

Alexandra Eisner,1* Gebhard Feierl,1 Gregor Gorkiewicz,1 Franz Dieber,2 Harald H. Kessler,1 Egon Marth,1 and Josef Köfer2

Institute of Hygiene, Medical University of Graz,1 Department of Veterinary Administration in Styria, A-8010 Graz, Austria2

Received 23 December 2004/ Accepted 10 May 2005


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ABSTRACT
 
Fecal samples from humans and food-producing animals were analyzed for the presence of vancomycin-resistant enterococci (VRE). The VRE carriage rate in humans was 6%, and there was a predominance of VanC-type resistance. Enterococcus faecium with vanA-mediated resistance was frequent in broiler chickens (42%) but rare in cattle and pig samples.


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INTRODUCTION
 
In 1986, the first human enterococcal isolates with glycopeptide resistance were detected in France and the United Kingdom (18, 25). Since then, vancomycin-resistant enterococci (VRE) have become a serious cause of nosocomial infections worldwide (12). Resistance to glycopeptides in enterococci is mediated by the vanA, vanB, vanC, vanD, vanE, or vanG gene cluster, and the vanA genotype is considered to be of major importance (3, 4, 9, 21). The vanA genotype is the predominant type of resistance reported in Europe; it is characterized by acquired inducible resistance to both vancomycin and teicoplanin and is transferable to other significant pathogens, like methicillin-resistant Staphylococcus aureus, due to its location on conjugative plasmids (10, 26). The vanB gene cluster confers inducible resistance to various levels of vancomycin, and isolates exhibit susceptibility to teicoplanin since this antibiotic is not an inducer. VanC-type glycopeptide resistance is characterized by chromosomally encoded and constitutively expressed resistance to low levels of vancomycin but susceptibility to teicoplanin. This resistance has been described as an intrinsic property of Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens (4). The remaining genotypes appear to occur infrequently among clinical isolates of VRE. In Europe, the use of the growth promoter avoparcin is thought to have selected for VRE in animal husbandry (1, 5, 16). Because VRE can be transferred from food animals to humans, the use of avoparcin was banned in all European countries in 1997. Consequently, several studies have shown that there has been a decline in the occurrence of VRE in fecal samples of food-producing animals, meat products, and healthy volunteers (6, 17, 24). In Austrian hospitals, the level of cases of human VRE infection or colonization remains low, and nosocomial cross transmission is considered a primary cause of acquisition of VRE (22).

The aim of the present study was to investigate the prevalence of VRE in southeast Austrian animal husbandry (cattle, pigs, and broilers) and in healthy volunteers. Moreover, the patterns of resistance of VRE obtained from animal husbandry to relevant clinical antimicrobials were determined.

A total of 619 animal fecal samples were obtained from different slaughterhouses; 208 cattle fecal samples originated from animals from 136 farms, 206 pig fecal samples originated from animals from 150 farms, and 205 broiler fecal samples originated from animals from 58 farms. Additionally, 200 human fecal specimens (127 females and 73 males; age range, 1 to 90 years) derived from nonhospitalized persons were investigated for the presence of VRE. None of the people had received antibiotic therapy during the previous 4 weeks.

One milliliter of a 10-fold-diluted fecal sample in 0.9% NaCl was added to 9 ml of Enterococcosel broth (BD Diagnostic Systems, Sparks, Md.) for enrichment. After incubation at 35°C for 24 h, 100 µl was subcultured onto vancomycin screen agar plates containing 6 mg vancomycin per liter (BD) and onto Colombia blood agar without vancomycin (BD) and incubated at 35°C for 24 h. Three colonies were randomly selected from each screening plate and subcultured on blood agar. All enterococcal isolates were identified on the basis of colony morphology, Gram staining, catalase and pyrrolidonyl arylamidase activities, Lancefield group D antigen, motility, yellow pigment production, and the 20 Strep and Rapid ID32 Strep API tests (bioMérieux, Marcy l'Etoile, France). The antibiotic susceptibility was determined with the VITEK 2 system (bioMérieux) using an AST-P524 test card (19). Additionally, MICs of vancomycin and teicoplanin were determined by the Etest method (AB Biodisk, Solna, Sweden), and the results were interpreted using the criteria recommended by the National Committee for Clinical Laboratory Standards (23). The glycopeptide resistance genotype was determined by PCR as described previously (11, 15).

The genotypes and species of VRE isolated from both animal and human sources are shown in Table 1. VRE were recovered from 45 of 208 (21.6%) cattle specimens, 48 of 206 (23.3%) pig specimens, and 158 of 205 (77.1%) broiler specimens. A total of 269 VRE were isolated from the animal fecal samples. Of these, 47 originated from cattle specimens, 49 originated from pig specimens, and 173 originated from broiler specimens. In total, 107 E. gallinarum strains and 63 E. casseliflavus strains were identified. As shown by PCR, all the E. gallinarum strains harbored the vanC1 gene, and all the E. casseliflavus strains harbored the vanC2 gene. No E. faecalis strains with glycopeptide resistance were detected. Ninety-nine VanA-type isolates were identified; these isolates included 88 Enterococcus faecium strains and 11 Enterococcus durans strains. All of these isolates expressed high-level vancomycin resistance (MICs, ≥256 mg/liter) and possessed the vanA gene, as confirmed by PCR. Except for one E. faecium isolate from cattle feces, all E. faecium vanA isolates originated from broiler fecal samples. The results of antibiotic susceptibility testing of the VRE isolates from animal sources obtained with the VITEK 2 system are shown in Table 2. High-level gentamicin resistance was seen in only two E. faecium isolates. Resistance to penicillin was found in 76% of the E. faecium isolates. The rates of resistance to ciprofloxacin and erythromycin differed for different Enterococcus species. High percentages of E. faecium (93%) and E. gallinarum (73%) isolates were found to be tetracycline resistant. Of the E. faecium isolates, 11% showed resistance or reduced susceptibility to quinupristin-dalfopristin. All VRE isolates were found to be susceptible to linezolid. Analysis of human stool samples revealed VRE carriage in 12 of 200 samples (6%). Nine E. gallinarum isolates, two E. casseliflavus isolates, and one E. faecium isolate with vanA-mediated resistance were found.


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  		TABLE 1. Species and genotypes of VRE strains isolated


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  		TABLE 2. Rates of antibiotic resistance for animal VRE isolates

Avoparcin was used as a growth promoter in broiler chicken, pig, and cattle husbandry in Austria, but it was banned 7 years ago. Surprisingly, we still found a high prevalence of VanA isolates in southeast Austrian poultry, whereas the prevalence in the pig and cattle specimens was low. We report here the first enterococcal isolates with high-level vancomycin resistance obtained from Austrian animal husbandry. Our results are in contrast to a recent study which reported the absence of VRE in food-producing animals (cows, pigs, and hens) in western Austria (22). The results of Mellmann et al., however, are not directly comparable to our results because the enrichment step was not included in their study. Incubation of specimens in enrichment broth was shown to be superior for isolation of VRE compared to direct isolation on vancomycin-supplemented plates (8). The reason for the high prevalence in southeast Austrian poultry remains unclear. Continuing high prevalence of E. faecium with VanA-type resistance despite discontinuation of the use of avoparcin has been described for Norwegian and Danish poultry farms (7, 13). E. faecalis with VanA-type resistance has also been shown to persist in the absence of avoparcin usage in New Zealand (20). The data indicate that the continuing occurrence of VRE in broiler flocks may be explained by persistence of VRE in the broiler house environment (14). In Danish pig herds, coselection by use of macrolides for therapy and growth promotion was described to be a mechanism for the persistence of glycopeptide resistance (2). In our study, only 28% of the vancomycin-resistant E. faecium isolates showed resistance to erythromycin, indicating that the linkage of resistance to macrolides and glycopeptides cannot be the only causative factor for the ongoing persistence. In Austria, tetracycline is a major antimicrobial used in broiler production, and the levels of resistance of VRE to this compound reflect this. Further investigations to clarify the possible coselection of VRE by tetracycline use are necessary. Little is known about the coselective capability of ionophore feed additives or metallic ions which might also select for VRE. Other potential mechanisms for the persistence include direct selective pressure by residues of avoparcin in the farm environment and transmission of resistant bacteria by eggs (7, 14). The high rate of resistance to penicillin in VanA-type E. faecium isolates from poultry investigated in the current study should be noted. Moreover, 11% of the vancomycin-resistant E. faecium strains were not susceptible to quinupristin-dalfopristin, suggesting that the use of the antimicrobial feed additive virginiamycin, although banned, has created a reservoir of streptogramin-resistant E. faecium in our study population. Limited data are available on the prevalence of human VRE colonization in Austria. A study in western Austria did not reveal any VRE carrier among 433 healthy volunteers (22). Similarly, one VanA-type VRE carrier was detected in our study, indicating that there is a minor VRE reservoir in the southeast Austrian community.

Although the number of cases of human VRE infections in Austrian hospitals is low, the agricultural VRE reservoir that still exists may threaten the community and human medicine because of the possible conjugative vanA gene transfer from enterococci to methicillin-resistant S. aureus, creating a dangerous pathogen that is difficult to treat (10, 22, 26). Surveillance of the VRE reservoir in animal husbandry and further investigations to clarify the mechanisms for persistence of VanA resistance are urgently required.


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ACKNOWLEDGMENTS
 
We thank Neil Woodford for a critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Hygiene, Medical University of Graz, Universitätsplatz 4, A-8010 Graz, Austria. Phone: 43(316)380-4383. Fax: 43(316)380-9648. E-mail: alexandra.eisner{at}meduni-graz.at. Back


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Applied and Environmental Microbiology, October 2005, p. 6407-6409, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6407-6409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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