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. 2008 Feb 1;74(6):1726–1730. doi: 10.1128/AEM.02164-07

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle▿

John F Anderson 1,†,‡, Torrey D Parrish 1,†, Mastura Akhtar 2, Ludek Zurek 2,3, Helmut Hirt 1,*
PMCID: PMC2268288  PMID: 18245252

Abstract

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10−6 per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.

Enterococci, in particular Enterococcus faecalis and Enterococcus faecium, are among the top three types of organisms isolated from nosocomial infections worldwide (15), and the prevalence of vancomycin-resistant enterococci has been on the rise in the last decades. Enterococci host a wide variety of mobile genetic elements and are considered a reservoir for acquisition and distribution of antibiotic resistance genes among gram-positive bacteria (5). The discussion about the use of antibiotics in animal husbandry and the potential for spread of resistant strains into the human community was highlighted by experiences in the European Union (EU) with the use of the growth promoter avoparcin. The link between avoparcin use and occurrence of vancomycin-resistant enterococci in the EU resulted in the eventual ban of avoparcin in animal feed in 1996 by Denmark, followed by an EU-wide ban in 1998. In the United States, over 50% of antibiotic use is estimated to occur in the animal industry (21). Consequently, antibiotic-resistant bacterial strains are routinely isolated from farm animals (12). While many studies have been conducted on antibiotic resistance among human populations and farm animals, less is known about the occurrence, distribution, and spread of antibiotic resistance in wild animal populations. It is expected that environments with no antibiotic pressure, such as nature preserves, harbor few antibiotic-resistant bacteria. Antibiotic resistance, however, could arise as consequence of inherent environmental pressure by antibiotic-producing bacteria or the spread of antibiotic-resistant bacteria to wild animal populations.

In this study, an American bison (Bison bison) population was chosen as the model system to evaluate antibiotic resistance in a free-living ruminant. We expected a significantly lower prevalence of antibiotic resistance in enterococci isolated from bison than in enterococci from a cattle population. The Konza Prairie research station is a 34.9-km2 native tall-grass prairie preserve located 10 km south of the city of Manhattan in the Northeast Kansas Flint Hills region. On a 9.5-km2 section, a 310- to 320-head-count herd of B. bison grazes on the native grasses of the prairie year round. The Konza bison population can serve as a sentinel for antibiotic pressure in the local environment. We found considerable resistance toward tetracycline and erythromycin in enterococci isolated from bison yet significantly less than in the cattle population. This is the first study of antibiotic resistance in enterococci associated with American bison. The results provide us with an opportunity to gain insight into the origin of antibiotic resistance and the flow of genetic elements between environments.

MATERIALS AND METHODS

Isolation of enterococci.

Fecal material was obtained by rectal sampling from 35 2-year-old bison during an annual spring roundup. Fecal samples were obtained at the same time from 20 cattle selected for a grazing study on the Konza Prairie. The material was placed in a sterile container on ice until arrival in the laboratory. The fecal material was suspended in 0.9% NaCl, and 100-μl aliquots were spread on mEnterococcus agar (Difco, Franklin Lakes, NJ) and incubated at 37°C for 48 h. Three to five distinct colonies were picked per individual bison or cattle sample for further characterization. Differences in results from the phenotypic analyses described below ensured that no duplicate strains were used. The affiliation of the isolates with the genus Enterococcus was confirmed by streaking them on bile-esculin agar (Difco).

Bacterial growth and culture conditions.

Enterococci were generally maintained and grown in Todd-Hewitt broth (THB; Difco) at 37°C. Solid media contained 1.5% agar. Blood-agar plates were made by adding a 5% volume of sterile bovine blood (Rockland, Gilbertsville, PA) to THB plates. Milk powder (1.5%) was added to THB plates to test for production of gelatinase.

Species determination.

The multiplex PCR approach of Kariyama et al. (14) was used to identify the species E. faecalis, E. faecium, Enterococcus gallinarum, and Enterococcus casseliflavus. Primers specific for vanB were used as suggested by Elsayed et al. (10). Species determination of isolates that either did not give a positive result or had ambiguous results in the multiplex PCR approach was performed by amplification and sequencing of the sodA gene for superoxide dismutase as previously described (27).

Antibiotic susceptibility testing and identification of antibiotic resistance determinants.

Susceptibility to antibiotics was evaluated on Mueller-Hinton agar (Difco) with standard antibiotic disks (Becton Dickinson, Franklin Lakes, NJ). Sensitivity and resistance were evaluated according to CLSI standards (24). The antibiotic resistance genes ermB, tetO, tetM, tetA, tetC, tetQ, tetS, and tetW were detected by PCR according to previously published protocols (25, 30, 33). A DNA fragment of 241 bp from the gyrA gene corresponding to the quinolone resistance-determining region was amplified with the 23-mer oligonucleotide primers 5′-CGG GAT GAA CGA ATT GGG TGT GA-3′ and 5′-AAT TTT ACT CAT ACG TGC TTC GG-3′, equivalent to nucleotide positions 150 to 172 and 368 to 390, respectively, of the Escherichia coli gyrA gene (16), and a 191-bp parC fragment was amplified with the 20-mer oligonucleotide primers 5′-AAT GAA TAA AGA TGG CAA TA-3′ and 5′-CGC CAT CCA TAC TTC CGT TG-3′ (positions 10 to 29 and 181 to 200, respectively, of the E. faecalis parC gene) (13). The two 23-mer oligodeoxyribonucleotides used as primers were 5′-CGG GAT GAA CGA ATT GGG TGT GA-3′ and 5′-AAT TTT ACT CAT ACG TGC TTC GG-3′.

Gelatinase activity, hemolysis, and bacteriocin.

Gelatinase activity was detected by a zone of clearance around the colonies on THB plates containing 1.5% milk powder. Isolates were tested for hemolysin production by streaking them on THB plates containing 5% cow blood. Bacteriocin activity was detected by applying Enterococcus isolates onto THB plates overlaid with 5 ml of THB soft agar (0.7%) inoculated with 50 μl of an overnight culture of test strain Staphylococcus aureus ATCC 27661, Streptococcus pyogenes ATCC 19615, or E. faecalis OG1RF (9).

Gene transfer assays.

Antibiotic resistance transfer was assayed using E. faecalis OG1RF as a recipient. Transfer on solid surfaces followed previously established protocols (35). High-frequency transfer in liquid culture was detected by coculturing of overnight cultures and incubation at 37°C. The culture was monitored for potential aggregate formation, indicating the presence of an aggregation substance. After 2 h, an aliquot of the culture was spread on plates containing rifampin at 200 μg/ml and the appropriate test antibiotic. To detect low-level gene transfer, potential donors and recipients were placed with a syringe on a 25-mm-diameter filter with 0.22-μm pores (Millipore, Billerica, MA). The donor- and recipient-covered filter was placed on nonselective THB plates and incubated overnight. The filter was removed the next morning, placed into 1 ml of THB, and vortexed and aliquots were placed on selective plates to determine transconjugants and recipient numbers.

Statistical analysis.

Statistical analysis of data was performed using InStat 3 software (GraphPad Software, Inc., San Diego, CA).

RESULTS

Species diversity in bison and cattle.

We isolated totals of 125 enterococcal colonies from the feces of 35 individual 2-year-old bison and 63 enterococcal colonies from 20 cattle. Enterococci were present in all sampled individuals. The concentrations of enterococci ranged from 102 to 107 CFU per gram of wet weight (mean of 2.8 × 106 CFU/g). In bison, E. casseliflavus was by far the most dominant species, with 62.4% of isolates (Table 1), and E. faecalis the second most prevalent, with 16% of isolates. Isolates from cattle showed a species distribution that was dominated by Enterococcus hirae (39.7%), followed by E. casseliflavus (28.6%). Only two isolates of E. faecalis (3.2%) and one isolate of E. faecium (1.6%) were obtained from cattle (Table 1).

TABLE 1.

Species distribution and antibiotic resistance phenotypes of enterococci in bison and cattlea

Species (no. of isolates/% of total isolates) No. (%) of isolates with resistance to indicated drug
Ciprofloxacin Erythromycin Tetracycline
E. casseliflavus
    Bison (78/62.4) 7 (9.0) 3 (3.8) 3 (3.8)
    Cattle (18/28.6) 6 (33.3) 0 2 (11.1)
E. hirae
    Bison (15/12) 1 (6.6) 1 (6.6) 3 (20)
    Cattle (25/39.7) 0 7 (28) 22 (88)
E. faecalis
    Bison (20/16) 7 (35) 0 3 (15)
    Cattle (2/3.2) 0 0 0
E. mundtii
    Bison (1/0.8) 0 0 0
    Cattle (15/23.8) 0 1 (6.7) 2 (13.3)
E. faecium
    Bison (3/2.4) 2 (75) 1 (33.3) 0
    Cattle (1/1.6) 0 0 0
Enterococcus spp.
    Bison (8/6.4) 1 (12.5) 0 1 (12.5)
    Cattle (2/3.2) 0 0 1 (50)
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a

Percentages of isolates with antibiotic resistance with reference to the total number of isolates of the species are given in parentheses.

Presence of hemolysins, gelatinase, and bacteriocins.

The majority (65.6%) of bison isolates showed hemolytic activity on bovine blood. The highest prevalence of hemolytic isolates was found in E. casseliflavus (89.7%). Secreted proteolytic activity (gelatinase) was found in 26.4% of all isolates and was mostly (70%) associated with E. faecalis (Fig. 1).

FIG. 1.

FIG. 1.

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Comparison of the hemolytic (A) and proteolytic (B) Enterococcus isolates in bison and cattle. Percentages of respective Enterococcus spp. in animal species are given. Only two isolates of E. faecalis were recovered from cattle.

Bison isolates (28%) showed the highest bacteriolytic activity against S. pyogenes (Table 2). E. casseliflavus dominated as a bacteriocin producer, with 42% of isolates showing activity against S. pyogenes. Four E. casseliflavus isolates showed activity against all three test organisms, four isolates showed activity against S. aureus and S. pyogenes, and five isolates inhibited E. faecalis and S. pyogenes. Interestingly, bacteriocin producers were significantly less abundant (P < 0.0004) in cattle isolates. A significant majority of bacteriocin producers (P < 0.0053) from cattle showed activity against S. aureus, in comparison to what was found for bison isolates, where the activity was more evenly directed against all test species.

TABLE 2.

Bacteriocin activitya

Species (% of bacteriocin-producing isolates) % of isolates with activity against indicated species
Enterococcus faecalis Staphylococcus aureus Streptococcus pyogenes
Bison
    All species (47.2) 18.4 19.2 28
    E. casseliflavus 21.8 26.9 42.3
Cattle
    All species (20.6) 1.5 17.4 3.1
    E. casseliflavus 50
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a

Bacterocin activity was assessed by zones of inhibition of the indicator organism surrounding Enterococcus isolates. Activities against indicated species are given as percentages of all isolates. The percentage of bacteriocin-producing isolates recovered from each animal species is given in parentheses.

Antibiotic resistance in enterococci isolated from bison and cattle feces.

A considerable number of enterococci isolated from bison were resistant to tetracycline (8%) and erythromycin (4%) (Fig. 2). One E. casseliflavus isolate was ampicillin resistant (MIC, 16 μg/ml), and one isolate of E. faecalis was found with low-level resistance (MIC, 4 μg/ml) to vancomycin. Tetracycline resistance was the leading antibiotic resistance phenotype in cattle (42.9%), followed by erythromycin resistance (12.7%). Overall antibiotic resistance in bison showed surprisingly high resistance to ciprofloxacin (14.4%). Ciprofloxacin resistance was found in only 9.5% of the cattle isolates. The incidence of tetracycline resistance in cattle was significantly higher (P < 0.0001 [Fisher's exact test]; 95% confidence interval, 31.3 to 56.9% for cattle and 2.2 to 13.4% for bison) than that in bison isolates. The difference in erythromycin resistance between Enterococcus isolates from cattle and bison was significant (P < 0.0347 [Fisher's exact test]; 95% confidence interval, 3.3 to 26.7% for cattle and 1.6 to 8.3% for bison). No significant difference in prevalence of ciprofloxacin resistance was found between bison and cattle (P < 0.4878). At the species level, ciprofloxacin resistance in bison was found in seven isolates each of E. casseliflavus and E. faecalis, whereas one E. hirae and one E. faecium isolate were ciprofloxacin resistant. All ciprofloxacin-resistant isolates in cattle were E. casseliflavus. Tetracycline resistance was found in three isolates each of E. casseliflavus, E. faecalis, and E. hirae in bison, whereas 88% of tetracycline-resistant cattle isolates were identified as E. hirae. Erythromycin resistance was found in seven isolates of E. hirae in cattle, with the remaining in Enterococcus mundtii.

FIG. 2.

FIG. 2.

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Overview of antibiotic-resistant Enterococcus spp. isolated from bison and cattle. Amp, ampicillin; Cip, ciprofloxacin; Cm, chloramphenicol; Erm, erythromycin; Gen, gentamicin; Tet, tetracycline; Van, vancomycin. *, P < 0.0347; **, P < 0.0001 in the abundance of antibiotic-resistant isolates in cattle and bison. The P value for ciprofloxacin was <0.4878.

Five multiple-antibiotic-resistant strains were isolated from bison, one E. casseliflavus isolate was resistant to three antibiotics (ciprofloxacin, erythromycin, and tetracycline), and one isolate each of E. casseliflavus (ciprofloxacin and erythromycin), E. hirae (erythromycin and tetracycline), E. faecalis (ciprofloxacin and tetracycline), and E. faecium (ciprofloxacin and erythromycin) was resistant to at least two antibiotics. Seven E. hirae isolates were found among the cattle isolates that exhibited resistance to erythromycin and tetracycline; one E. mundtii isolate was also resistant to those two antibiotics. The prevalences of multiple-antibiotic-resistant isolates were thus 4% in bison isolates and 12.7% in cattle, indicating a significant difference between the two populations (P < 0.0347, Fisher's exact test).

Molecular analysis of antibiotic resistance genes.

PCR analysis identified all erythromycin resistance genes as ermB. All tetracycline resistance genes from cattle isolates belonged to the tetO class of resistance genes. Although the majority of tetracycline resistance genes in the bison isolates were also identified as tetO, two E. faecalis isolates carried tetM. These two isolates also tested positive for the ermB gene but phenotypically showed only intermediate erythromycin resistance. The unexpected and relatively high prevalence of ciprofloxacin resistance, especially in bison, prompted us to examine the molecular mechanism of the resistance. Sequencing of gyrA and parC of selected isolates of both E. casseliflavus and E. faecalis revealed no amino acid changes in the conserved regions of GyrA and ParC.

Transfer of antibiotic resistance genes.

The tetracycline resistance found in the 10 bison isolates was transferred from all isolates to OG1RF on solid media and from 7 isolates in liquid media. The transfer frequency of tetracycline resistance in liquid media averaged 8.1·10−6 (±9.3·10−6). Similarly, all 27 tetracycline resistance determinants from cattle isolates transferred to OG1RF on solid media, and 24 (89%) did so in liquid media. Transfer frequency was comparable (4.4·10−6 ± 5.2·10−6). The transfer frequencies for tetracycline were consistent with the presence of conjugative plasmids. Erythromycin resistance was not transferred in liquid medium or on a solid surface from enterococci obtained from bison or cattle.

DISCUSSION

Few studies that investigate the spread of antibiotic resistance genes in wild animals are available. It is unclear whether antibiotic pressure exists in natural environments or antibiotic-resistant strains selected by human activity spread to wild animal populations. A survey of Enterobacteriaceae from a wide diversity of mammal and marsupial species in Australia (28) detected a remarkable prevalence of antibiotic resistance even in locations far removed from human influence, supporting the first notion. However, a study of antibiotic resistance in enterococci isolated from 20 different species of wild animals in Portugal showed resistance in 28 and 20% of isolates to tetracycline and erythromycin, respectively (26). This result would support a spillover effect due to heavy use of antibiotics in EU agriculture before the ban of several antibiotics as growth promoters in 1997. Supporting this hypothesis is a recent study characterizing Escherichia coli isolates from environments with various levels of exposure to human population density (29). The data suggest a strong correlation between human population density and the spread of antibiotic resistance to wildlife.

Enterococci are associated with a wide variety of antibiotic resistance determinants that are often found on mobile genetic elements (23). Enterococci are ubiquitous in the intestinal tract of mammals and can be found associated with grasses and isolated from flies (18, 22). Their frequent association with mobile genetic elements and their ubiquity make enterococci the ideal reservoir for antibiotic resistance genes, mediating between different environments and bacterial species.

We found low prevalences of the two clinically important species E. faecalis and E. faecium (4.8% combined) in our cattle isolates, consistent with the reported low abundance in cattle (6, 32). A significant difference was found in species distribution of enterococci isolated from bison. E. casseliflavus was by far the dominant species, and interestingly, E. faecalis was the second most common species. E. casseliflavus is often associated with water and plants (22) yet can become the dominant Enterococcus species in the human intestinal tract with constant exposure (11). The abundance of E. casseliflavus among bison isolates could therefore be caused by a constant supply of plant-associated E. casseliflavus by grazing. The relatively high prevalence of E. faecalis in bison is surprising. It is conceivable that E. faecalis is better suited for colonizing bison or that differences between the diet of cattle and the native grass diet of bison on the Konza Prairie favor E. faecalis.

Whereas the detection of ermB as a major resistance determinant for this class of antibiotics in bovine isolates is no surprise (7), the detection of tetM in two E. faecalis isolates obtained from bison was unexpected. tetM is commonly associated with human isolates, whereas several recent studies of Streptococcus agalactiae confirm the prevalence of tetO in cattle in Brazil and the United States (7, 8). Both groups found tetM restricted to human clinical isolates, where tetO plays only a minor role.

The main objective of this study was to compare the prevalences of antibiotic resistance determinants in the two ruminant populations. Not surprisingly, levels of resistance to tetracycline and erythromycin were higher in enterococci from cattle, evidence for the selective pressure placed on domestic animals by the use of these antibiotic classes in veterinary therapy and for growth promotion. Although the frequencies of tetracycline (8%) and erythromycin (4%) resistance in bison appear relatively low, if no antibiotic pressure or no transfer of antibiotic resistance genes from an outside environment occurs, one should expect lower antibiotic resistance rates.

Enterococci commonly carry mobile genetic elements and due to their ubiquity in many environments are suspected to serve a role as accumulators and distributors of antibiotic resistance determinants. The most notorious cases for the spread of antibiotic resistance genes were the transfer of vanA from E. faecalis to S. aureus in several unrelated instances (1, 4, 34). All tetracycline resistance elements expressed showed transfer to E. faecalis in mating assays, with 90% of the isolates in bison able to transfer tetracycline resistance in a liquid mating. The observed transfer frequency in liquid of 10−6 lies in the range of conjugative plasmids such as pAMβ 1 and pIP501 (20). One of the bison isolates showed tetracycline resistance transferring at a rate of 10−8 on solid media only, indicating that transfer could be based on a conjugative transposon. Erythromycin resistance based on ermB was not associated with mobility. This was somewhat surprising since ermB is usually associated with plasmids and transposons (2, 31).

Remarkably, no difference in level of ciprofloxacin resistance could be seen between cattle and bison, and the prevalence in bison was higher than that in cattle, although the difference was not significant. The overall prevalence of ciprofloxacin resistance in our cattle isolates was low in comparison to what was found in recent studies that reported ciprofloxacin resistance levels of 55% and 47% in beef and dairy cattle, respectively (12). No sequence alterations in GyrA or ParC were detected, which suggests that ciprofloxacin resistance could be based on a nonspecific efflux mechanism (17).

The surprisingly high incidence of antibiotic resistance in bison on the Konza Prairie raises the question of where the antibiotic-resistant strains originated. The herd is under veterinary control, and medical records show that none of the animals had been exposed to antibiotics since the establishment of the herd in 1988. A clinical study with oral streptococci demonstrated a rapid selection of antibiotic-resistant strains and persistence over the background level for at least 180 days after the last antibiotic administration (19). For the Konza bison population, this would indicate a one-time high exposure with levels returning to background levels, a constant exposure to antibiotics from an as-yet-uncharacterized source, or reacquisition of antibiotic-resistant bacterial strains from the surrounding environment. A potential explanation for the high incidence of antibiotic resistance found in the Konza bison population could be the transfer of antibiotic-resistant enterococci by flies. The surrounding farms lie well within the flying range of houseflies, which are capable of carrying high concentrations of enterococci per fly (17). Fly development takes place in the manure of cattle, and intestinal flora of cattle can easily be detected in the gut of the insect (36). The range of houseflies has been estimated at 3 miles, with relocations by steady directional winds of up to 10 miles (3). A recent study of flies isolated from restaurants in a town in Kansas revealed a sizable antibiotic resistance gene pool in enterococci isolated from houseflies (18). The source of water for the animals is King's creek, originating on the Konza Prairie. The water source is therefore an unlikely origin of resistant enterococci. Other potential sources are birds and larger mammals, such as deer, that could transfer antibiotic-resistant strains from outside environments into the prairie preserve.

The bison population on the Konza Prairie provides an opportunity to monitor the impact of antibiotic use on the natural environment and can serve as a sentinel for antibiotic pressure from the surrounding farm and urban environments. The low but significant presence of antibiotic resistance genes in bison will allow tracking of the flow of resistance genes and the identification of the origins of these determinants in the environment.

Acknowledgments

This work was supported by USDA grant no. 2005-35302-16340 to Ludek Zurek and Helmut Hirt. John Anderson was a Star Trainee in the Kansas INBRE program funded by P20 RR016475.

Special thanks to Gene Townsend and Tom van Slyke for access to the animals on the Konza Prairie. We thank Alicia Casey for technical assistance and Lynn Hancock for critical reading of the manuscript.

This paper is contribution no. 08-211-J from the Kansas Agricultural Experiment Station.

Footnotes

▿

Published ahead of print on 1 February 2008.

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