Evaluation of Chromocult enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers

AIMS: To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult enterococci agar) recently developed by Merck. METHODS AND RESULTS: The study was carried out comparing Chromocult enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed. CONCLUSIONS: A good sensitivity of 98% was obtained for Chromocult agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P<0.01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult enterococci agar. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.     

Comparison of pulsed-field gel electrophoresis patterns of antimicrobial-resistant Escherichia coli and enterococci isolates from the feces of livestock and livestock farmers in Japan

Seven hundred thirty-nine animal strains and 662 livestock-farmer strains, consisting of Escherichia coli and enterococci, were examined for their pulsed-field gel electrophoresis (PFGE) and antimicrobial-resistance patterns. Two hundred fifty-eight and 203 PFGE patterns were found among 739 animal strains isolated from animals comprising broilers, pigs and cattle, and 662 human strains isolated from livestock farmers, respectively, from 27 farms in Japan. These results demonstrated that the PFGE patterns found among E. coli and enterococci strains from animals and livestock-farmers were heterogeneous and considerably diverse. The strains having both the identical PFGE pattern and the same drug-resistance pattern were defined as a single clone in this study. Seven types of E. coli and enterococci clones were shared among animals within the same farms and between the different farms housing the same animal species. The 25 strains (3.4%) of 739 E. coli and enterococci animal strains belonged to these seven types of clones. Only three types of E. coli clones were shared among animals between the different farms housing different animal species, but no identical E. faecalis or E. faecium clones were found between different animal species farms. The 15 strains (2.0%) of 739 E. coli and enterococci animal strains belonged to these three types of clones. Additionally, the 11 strains (1.5%) of 739 E. coli and enterococci strains isolated from animals were identical clones to strains isolated from livestock farmers of the same farm. These results suggest that the transmission of animal clones to livestock farmers or vice versa is less common.     

Environmental contamination by vancomycin resistant enterococci (VRE) in Swedish broiler production

Background  

Vancomycin resistant enterococci are a frequent cause of nosocomial infections and their presence among farm animals is unwanted. Using media supplemented with vancomycin an increase in the proportion of samples from Swedish broilers positive for vancomycin resistant enterococci has been detected. The situation at farm level is largely unknown. The aims of this study were to obtain baseline knowledge about environmental contamination with vancomycin resistant enterococci in Swedish broiler production and the association between environmental contamination and colonisation of birds.     

Is the iron an essential nutrient for enterococci?

Enterococci were considered as not requiring iron. The aim of study was evaluation of relationship between enterococci and iron. This study examined these relationships in a 71 strains belonging to two species--Enterococcus faecalis and Enterococcus faecium, which are often isolated from human infections. The iron is an essential nutrient for enterococci. Demonstrated that iron--regardless of the concentration in the medium--is collected during growth. Iron deficiency in the nutrient medium resulted in changes in the kinetics of growth of enterococci. Inhibiting the growth of enterococci by iron chelators and lack of inhibition are further proof of this demand for iron bacteria. Enterococci have the ability to acquire this important element of its connections with natural and synthetic chetators with different strength of chemical bonding and structure. Bacteria of the genus Enterococcus have a natural resistance to many antimicrobial agents. In the hospital environment can easily acquire resistance genes to many other classes of antimicrobial compounds. For these reasons, treatment of enterococal infections poses more difficulties. Inhibition of iron uptake in enterococci can be helpful in reducing and combating enterococal infections.     

Comparison of fluorescent gentamicin-thallous-carbonate and KF streptococcal agars to enumerate enterococci and fecal streptococci in meats.

Two selective and differential media were compared for their abilities to enumerate enterococci and fecal streptococci in pork, beef, and poultry products. Counts obtained on KF streptococcal (KF) agar were compared with counts obtained on fluorescent gentamicin-thallous-carbonate (fGTC) agar. Reactions of 13 known enterococcal species were also observed. All 13 species of enterococci as well as Streptococcus bovis and Streptococcus equinus grew equally well on fGTC agar. KF streptococcal medium allowed growth of most species of enterococci but not S. bovis and S. equinus. Quantitative comparisons between the two media inoculated with pure cultures of known species of enterococci revealed equivalent plate counts following incubation. However, when meat samples were plated, counts on fGTC agar were consistently and significantly higher than counts on KF agar for all sample sources.     

Spatial and Temporal Variation in Enterococcal Abundance and Its Relationship to the Microbial Community in Hawaii Beach Sand and Water

Recent studies have reported high levels of fecal indicator enterococci in marine beach sand. This study aimed to determine the spatial and temporal variation of enterococcal abundance and to evaluate its relationships with microbial community parameters in Hawaii beach sand and water. Sampling at 23 beaches on the Island of Oahu detected higher levels of enterococci in beach foreshore sand than in beach water on a mass unit basis. Subsequent 8-week consecutive samplings at two selected beaches (Waialae and Kualoa) consistently detected significantly higher levels of enterococci in backshore sand than in foreshore/nearshore sand and beach water. Comparison between the abundance of enterococci and the microbial communities showed that enterococci correlated significantly with total Vibrio in all beach zones but less significantly with total bacterial density and Escherichia coli. Samples from the different zones of Waialae beach were sequenced by 16S rRNA gene pyrosequencing to determine the microbial community structure and diversity. The backshore sand had a significantly more diverse community and contained different major bacterial populations than the other beach zones, which corresponded to the spatial distribution pattern of enterococcal abundance. Taken together, multiple lines of evidence support the possibility of enterococci as autochthonous members of the microbial community in Hawaii beach sand.     

A collaborative study of a method for enumeration of probiotic enterococci in animal feed

AIMS: Validation of an enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic enterococci used as feed additives. METHODS AND RESULTS: Twenty laboratories in 12 European countries carried out a collaborative study. A plate count method using bile esculin azide (BEA) agar was used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Enterococci were present in the samples as a single component or in mixtures with other probiotic feed additives. The enumeration of enterococci on BEA agar showed a relative standard deviation (RSD)r of 1.5-3.6% and an RSD(R) between 2.9 and 7.4%. BEA agar was selective for enterococci in the presence of other probiotic micro-organisms such as pediococci, lactobacilli and yeast. CONCLUSIONS: For routine analysis of viable enterococci concentrations in feeding stuffs, the use of BEA is recommended. This methodology is not applicable for mineral feeds. SIGNIFICANCE AND IMPACT OF THE STUDY: An official control method for enumeration of authorized probiotic enterococci in feeding stuffs was validated. The results are intended for consideration for adoption as CEN and ISO standards.     

Evidence of septic system failure determined by a bacterial biochemical fingerprinting method

AIMS: To provide evidence of septic system failure by comparing two faecal indicator bacteria, enterococci and Escherichia coli, from defective septic tanks and adjacent creeks. METHODS AND RESULTS: A biochemical fingerprinting method was used to type and compare enterococci and E. coli strains from 39 septic tanks with creek water samples. Phenotypic diversity of enterococci (0.5 +/- 0.3) and E. coli (0.5 +/- 0.3) in septic tanks were significantly lower than those found in water samples (0.8 +/- 0.1, P < 0.0001 for enterococci and 0.9 +/- 0.1, P < 0.0001 for E. coli). Among 1072 enterococci isolates tested from septic tanks, 203 biochemical phenotypes (BPTs) were found of which 98 BPTs from 33 septic tanks were identical to several water samples. Similarly, among 621 E. coli isolates tested from septic tanks, 159 BPTs were found of which 53 BPTs from 26 septic tanks were also identical to water samples. The number of the latter bacteria was significantly (P = 0.01) higher in water samples collected from downstream compared with that of upstream in the study area. A high similarity between the populations of both indicator bacteria was also found between defective septic tanks and downstream water samples further indicating the contamination of both creeks by defective septic systems. CONCLUSIONS: Biochemical fingerprinting of faecal indicator bacteria is a useful and rapid method to provide direct evidence for septic system failure. Combination of both faecal indicator bacteria (enterococci and E. coli) provides a better judgement of the performance of a septic system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to provide direct evidence of septic system failure by identifying the presence of specific bacterial types in septic tanks and surface waters. Based on our findings, we suggest that the performance evaluation of a septic system should be accompanied by direct analysis of faecal indicator bacteria.     

Enterococci as a cause of infectious complications after surgery

Self-obtained data about study of enterococci as etiologic agents of infectious complications after cardiovascular and transplantation surgery are presented in the article. 465 strains of enterococci were isolated from different biosubstrates. Isolation rate of different species of enterococci in blood, urine, fromwounds, trachea, cardiacvalves, as well as in the environment and on wear of healthcare workers were determined. Most frequently enterococci were isolated from urine (47.7% of cases) and from trachea (24.3% of cases). Enterococcal bacteremia was determined in 9.7% of cases and represented a significant part in Grampositive microflora--38.5% of all isolates. High resistance of enterococci to antibiotics and well lysis by phages were noted.     

The relationships between salmonellas and faecal indicator concentrations in two pools in the Australian wet/dry tropics.

The relationships between total coliform, faecal coliform, enterococci and salmonella concentrations were investigated at Berry and Howard Pools in the Australian wet/dry tropics. Both pools have catchments with minimal human activity and no major point source of faecal pollution. Forty-five indicator and salmonella enumerations were made from each pool over a 1 year period. Salmonellas were isolated from 69% and 96% of samples collected from Howard and Berry Pools respectively, the maximum (MPN) concentration was 110/100 ml. Native fauna were the primary salmonella source. Spearman rank correlations between indicator organisms and salmonella at Howard Pool were significant at the 5% level and approximated 0.6. At Berry Pool, total coliform and enterococci Spearman rank correlations with salmonella were also statistically significant, approximating 0.3; faecal coliforms and salmonella rankings, however, were unrelated. The higher correlation coefficients at Howard Pool were attributed to its small catchment (4 km2) and the more recent nature of faecal contamination compared with Berry Pool which has a catchment of 130 km2. The results highlight the spatial variability of the indicator/pathogen numerical relationship. Total coliform and enterococci counts, as indicators of faecal pollution, were similar and more consistent than faecal coliforms.     

Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14C (mean river water temperature). Sunlight inactivation (k(S)) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k(D)) rates in enclosed (control) chambers. The overall k(S) ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms greater-than-or-equal E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Trimethoprim resistance in enterococci: microbiological and biochemical aspects

Synergy was found between sulphonamide and trimethoprim in ratios 1:1 and 20:1 against both trimethoprim-sensitive enterococci (17 strains) and trimethoprim-resistant enterococci (23 strains). Many of these strains were resistant to kanamycin, tetracycline, streptomycin and/or erythromycin. Resistance to kanamycin, but not to trimethoprim, was clearly associated with the presence of a plasmid of molecular weight 35-45 Md. Elimination of this plasmid in three out of four highly trimethoprim resistant strains brought about loss of resistance to both kanamycin and trimethoprim. Furthermore, it was possible to transfer trimethoprim resistance from three of five highly resistant strains, but not from three strains with low-grade resistance. It is concluded that resistance to trimethoprim in enterococci can be encoded on a plasmid, and that the gene responsible may be on a transposon. No significant differences were found between the specific activities of dihydrofolate reductase from trimethoprim-sensitive and -resistant strains. The enzyme from resistant strains was several orders of magnitude less susceptible to inhibition by trimethoprim than was the enzyme from sensitive strains.     

Distribution and persistence of fecal bacterial populations in liquid and dewatered sludge from a biological treatment plant

The changes in composition and structure of fecal coliforms (FC) and enterococci (ENT) populations, as well as the elimination of spores of sulphite-reducing bacteria (SRB), were compared between municipal sewage and their derived sludge in a biological treatment plant in order to determine any selective reduction or adsorption to sludge during the treatment process. Additionally, the persistence of antibiotic-resistant enterococcal populations in two kinds of sludge was also considered to evaluate their potential elimination in the treatment process. Microbial indicators, vancomycin-resistant and erythromycin-resistant enterococci were enumerated. The structure and composition of FC and ENT populations were determined by biochemical fingerprinting and clustering analyses. Raw and treated sewage showed a concentration of FC 1 log unit higher than ENT and nearly 2 log units higher than spores of SRB. However, the three studied indicators showed similar concentrations in both types of sludge. Consequently, FC were eliminated in higher proportion than ENT and spores of SRB in sludge. FC and ENT populations showed high diversity and similarity population indexes for all kinds of samples. Antibiotic-resistant enterococci persisted in a similar proportion in respect to total enterococci not only in treated sewage but also in sludge. The persistence of antibiotic-resistant strains in sludge as well as in treated sewage should be considered if they are used for land disposal or for water reutilization, respectively.     

Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan

Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied. This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E. coli and enterococci during the summer of 2002. Both E. coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (+/- standard errors) of 5.3 (+/- 4.8) and 4.8 (+/- 4.5) per g (dry weight). E. coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R(2) = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16). Both E. coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4 degrees C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E. coli and enterococci as indicators of water quality for freshwater recreational beaches.     

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.     

正常人肠道肠球菌耐药性与生物膜形成关系的初探

目的了解正常人肠道肠球菌对临床常用抗生素的耐药水平和其生物膜的形成情况,并初步探讨肠球菌的耐药性与其生物膜形成之间的关系。方法用K-B法测定正常人肠道肠球菌对15种抗生素的敏感性,用96孔聚苯乙烯板进行生物膜形成试验。结果生物膜形成阳性菌株对高浓度链霉素、四环素和红霉素的耐药性(耐药率分别为42.9%、90.5%、71.4%)显著高于生物膜形成阴性的菌株(耐药率分别为4.8%、38.1%、42.8%),对其余12种抗生素的耐药性与生物膜形成阴性株差异无统计学意义。结论生物膜形成对肠球菌耐药性增强有一定作用,但还与其本身耐药性和抗生素的性质有关。     

A Clinical Study of the Role of Enterococci as Sole Agents of Wound and Tissue Infection

Patients who had enterococci isolated from wounds or tissues were identified from laboratory records. The charts of patients with pure cultures of enterococci were reviewed to determine the degree of clinically significant infection. We found that the frequency of infections in patients with pure cultures of enterococci was not significantly different from the frequency of infections in a control series of patients with negative cultures, but that it was significantly different from the frequency of infections in a series of patients with pure cultures of Staphylococcus aureus. Our conclusion that enterococci are not by themselves significant pathogens in wound or tissue infections is supported by a few experimental studies of other authors.     

Enterococci as indicators of Lake Michigan recreational water quality: comparison of two methodologies and their impacts on public health regulatory events

The frequency of poor-water-quality advisories issued in Milwaukee and Racine, Wisconsin, in the absence of identifiable sources of contamination brought into question the reliability of the present indicator organism, Escherichia coli. Enteroccoci have been suggested as an alternative to E. coli for freshwater monitoring due to their direct correlation to swimmer-associated gastroenteritis. The purpose of this research was threefold: (i) to explore enterococci as an alternative to E. coli for monitoring freshwater Lake Michigan beaches, (ii) to evaluate the impact of the two indicators on regulatory decisions, and (iii) to compare membrane filtration m-enterococcus agar with indoxyl-beta-D-glucoside to a chemical substrate technique (Enterolert) for the recovery of enterococci. Recreational water samples from Milwaukee (n = 305) and Racine (n = 153) were analyzed for the enumeration of E. coli and enterococci using IDEXX Colilert-18 and Enterolert. Correlation between the indicators was low (R(2) = 0.60 and 0.69). Based on U.S. Environmental Protection Agency bacterial indicator threshold levels of risk for full body immersion, using enterococci would have resulted in 56 additional unsafe-recreational-water-quality advisories compared to the total from using E. coli and the substrate-based methods. A comparison of the two enterococcal methods (n = 124) yielded similar results (R(2) = 0.62). This was further confounded by the frequent inability to verify enterococci from those wells producing fluorescence by the defined substrate test using conventional microbiological methods. These results suggest that further research is necessary regarding the use of defined substrate technology interchangeably with the U.S. Environmental Protection Agency-approved membrane filtration test for the detection of enterococci from fresh surface water.     

Host species-specific metabolic fingerprint database for enterococci and Escherichia coli and its application to identify sources of fecal contamination in surface waters

A metabolic fingerprint database of enterococci and Escherichia coli from 10 host groups of animals was developed to trace the sources of fecal contamination in surface waters. In all, 526 biochemical phenotypes (BPTs) of enterococci and 530 E. coli BPTs were obtained from 4,057 enterococci and 3,728 E. coli isolates tested. Of these, 231 Enterococcus BPTs and 257 E. coli BPTs were found in multiple host groups. The remaining 295 Enterococcus BPTs and 273 E. coli BPTs were unique to individual host groups. The database was used to trace the sources of fecal contamination in a local creek. The mean diversities (Di) of enterococci (Di = 0.76 +/- 0.05) and E. coli (Di = 0.88 +/- 0.04) were high (maximum 1) in water samples, indicating diverse sources of fecal contamination. Overall, 71% of BPTs of enterococci and 67% of E. coli BPTs from water samples were identified as human and animal sources. Altogether, 248 Enterococcus BPTs and 282 E. coli BPTs were found in water samples. Among enterococci, 26 (10%) BPTs were identical to those of humans and 152 BPTs (61%) were identical to those of animals (animal BPTs). Among E. coli isolates, 36 (13%) BPTs were identical to those of humans and 151 (54%) BPTs were identical to those of animals. Of the animal BPTs, 101 (66%) Enterococcus BPTs and 93 (62%) E. coli BPTs were also unique to individual animal groups. On the basis of these unique Enterococcus BPTs, chickens contributed 14% of contamination, followed by humans (10%), dogs (7%), and horses (6%). For E. coli, humans contributed 13% of contamination, followed by ducks (9%), cattle (7%), and chickens (6%). The developed metabolic fingerprint database was able to distinguish between human and animal sources as well as among animal species in the studied catchment.     

Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ～194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.