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¹ Department of Biological and Allied Health Sciences, Fairleigh Dickinson University, Madison, New Jersey 07940, USA
² Corresponding author (email: junem{at}fdu.edu)

	ABSTRACT

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ABSTRACT:   Thermotolerant fecal indicator organisms carried by migratory waterfowl may serve as reservoirs of antibiotic resistance. To determine the extent to which such antibiotic resistance markers were present in migratory Canada geese (Branta canadensis) on the Maryland Eastern Shore, we isolated Enterococcus spp. and Escherichia coli from fresh feces and examined the antibiotic resistance profiles of these bacteria. Samples were obtained in October 2002, January 2003, and March 2003. Thermotolerant E. coli counts ranged from 0 to 1.0x10⁷ colony forming units (CFU)/0.1g (g^–1) wet weight of feces, whereas Enterococcus spp. counts ranged from 1.0x10²–1.0x10⁷ CFU g^–1 wet weight of feces. Primary isolates of each indicator organism were tested against a panel of 10 antibiotics. Greater than 95% of E. coli isolates were resistant to penicillin G, ampicillin, cephalothin, and sulfathiazole; no E. coli were resistant to ciprofloxacin. Enterococcal isolates showed highest resistance to cephalothin, streptomycin, and sulfathiazole; no enterococci were resistant to chloramphenicol. The tetracyclines, streptomycin, and gentamycin provided the greatest discrimination among E. coli isolates; chlortetracycline, cephalothin, and gentamycin resistance patterns provided the greatest discrimination between enterococcal strains. Multiple antibiotic resistance (MAR) profiles were calculated: fall (E. coli = 0.499; enterococci = 0.234), winter (E. coli = 0.487; enterococci = 0.389), and spring (E. coli = 0.489; enterococci = 0.348). E. faecalis and E. faecium, which are recognized human nosocomial pathogens, were cultured from winter (44 and 56%, respectively) and spring (13 and 31%, respectively) fecal samples.
  Key words:  Branta canadensis, Canada goose, Enterococcus spp., Escherichia coli, multiple antibiotic resistance.

	INTRODUCTION

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Antibiotics have become commonplace in our environment (Col and O’Connor, 1987). They are widely used in medical therapy, animal husbandry, and agriculture (Houndt and Ochman, 2000; Vidaver, 2002). Microbes may develop resistance to antibiotics under selective pressure, or they may acquire antibiotic resistance determinants without direct exposure to an antibiotic (Koshland, 1994). Most antibiotic resistance genes reside on horizontally mobile elements (HMEs). These HMEs, which include viruses, conjugative plasmids, integrons, and transposons, can readily transfer antibiotic resistance genes from one organism to another (Heinemann, 1998), and can persist in bacterial genomes in the absence of selective pressure by antibiotics (Jabes et al., 1989).

The thermotolerant bacteria Escherichia coli and Enterococcus spp. are routinely used by public health authorities as indicators of fecal pollution in recreational waters (United States Environmental Protection Agency, 1986). These fecal indicator organisms are found in the digestive tracts of many homeothermic animals, and E. faecalis and E. faecium are potential nosocomial pathogens. Conjugative exchange of antibiotic resistance plasmids in E. coli from migratory waterfowl other than Canada geese (Branta canadensis) has been demonstrated (Tsubokurea et al., 1995). Genetic exchange of virulence determinants had been shown to occur between food and medical enterococcal isolates (Eaton and Gasson, 2001).

We were interested in determining whether the thermotolerant fecal indicator organisms carried by migratory Canada geese might serve as reservoirs of antibiotic resistance. Because these migratory waterfowl have a large flight range, it is possible that they may be effective disseminators of antibiotic resistance determinates. To determine the extent to which these geese carry bacteria with antibiotic resistance markers, we assessed the prevalence and combinations of antibiotic resistance determinants in the fecal indicator organisms of migratory Canada geese.

Several researchers have enumerated fecal E. coli from Canada geese (Alderisio and DeLuca, 1999; Kullas et al., 2002), but little is known about the fecal levels of enterococci in these birds. Therefore, we also evaluated levels of the thermotolerant E. coli and Enterococcus spp. present in feces from these birds.

	MATERIALS AND METHODS

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Sample collection

Goose feces were collected from winter wheat or soybean fields utilized by migratory Canada geese on the Oxford peninsula on Maryland’s Eastern Shore (38°69'N, 76°14'W); all samples were collected within a 1.6-km radius. Samples were collected along a single transect through large flocks of geese (n>200) to provide maximum assurance that each sample was from a different individual bird. Samples were collected in late October 2002 (fall), late January 2003 (winter), and early March 2003 (spring); air temperatures at sampling were 8.8 C, 0.3 C, and 8.8 C, respectively.

Fresh goose feces (within 5 min of defecation) were collected from the ground into an everted sterile sampling bag (Fisher Scientific, Pittsburgh, Pennsylvania, USA) placed over the hand of the collector. Care was taken to collect only the fresh fecal sample, avoiding soil and grass contaminants.

Microbial enumeration and identification

All samples (fall, n = 21; winter, n = 25; spring, n = 17) were processed within 4 hr. Each sample was weighed, diluted 1:10 (w/v) in phosphate buffered saline (pH 7.0), shaken vigorously for 3–5 min, and then placed on a platform shaker at 150 rpm for 30 min. After these steps, the sample was allowed to stand for 10 min so that solids could settle. Serial dilutions were made in phosphate buffered saline, and dilutions were spread plated on Levine Eosin Methylene Blue Agar (EMB) and on Enterococcosel Agar (ENT) (BBL^®, Becton, Dickinson, and Company, Sparks, Maryland, USA). Plates were incubated at 44.5 C for 24–48 hr, and characteristic colonies (E. coli on EMB; enterococci on ENT) were counted. Samples exhibiting no growth on spread plates were streak plated from the original 1:10 dilution, which had been held at 24 C overnight, to detect recovery of stressed cells.

All thermotolerant E. coli were verified by growth at 44.5 C and o-Nitrophenyl-ß-D-gal-actopyranoside/methylumbelliferyl-ß-D-glucuronide positive reactions (Colilert^®, Idexx, Westbrook, Maine, USA). Thermotolerant enterococci were verified by lack of catalase production, esculin hydrolysis, and growth at 44.5 C. Species identification of selected nonpigmented enterococci from winter and spring samples was performed according to the method of Manero and Blanch (1999).

Antibiotic resistance testing

Colonies derived from primary spread plates were used to assess antibiotic resistance patterns. Isolated colonies of each species were picked from the EMB or ENT isolation plates into sterile 96-well microtiter plates containing 180 µl of M-FC broth (for E. coli isolation) or Enterococcosel broth (for isolation of enterococci) (both BBL^®, Becton, Dickinson, and Company, Sparks, Maryland, USA) and incubated at 44.5 C for 24–48 hr. A maximum of 24 individual colonies from each positive E. coli and enterococci sample were picked, but for some samples, fewer than 24 distinct colonies were present. Only samples containing more than six colonies of either genus were evaluated. All isolates (E. coli: n = 447, 264, 211; enterococci: n = 367, 542, 355) from fall, winter, and spring samples, respectively, were replica-plated onto antibiotic and control plates and incubated at 37 C for 48–72 hr (Wiggins, 1996). Each isolate was tested against a panel of 10 antibiotics on Trypticase Soy Agar (BBL^®) plates supplemented with tetracycline (ICN Biochemicals, Aurora, Ohio, USA), chlortetracycline hydrochloride (ICN Biochemicals), cephalothin (Sigma-Aldrich, St. Louis, Missouri, USA) (each at 25 µg/ml), ampicillin (Sigma), streptomycin (Fisher Scientific, Fair Lawn, New Jersey, USA), gentamycin (Fisher Scientific) (each at 10 µg/ml), ciprofloxacin (ICN Biochemicals) (5 µg/ml), or sulfathiazole (ICN Biochemicals) (200 µg/ml); control plates were Trypticase Soy Agar. The antibiotic panel was chosen to include antibiotics with potential efficacy against both E. coli and the enterococci. The antibiotic concentrations used were those that have been shown to allow discrimination between isolates on the basis of susceptibility patterns differences (Kaspar et al., 1990; Wiggins, 1996) or to correspond to concentrations used in clinical in vitro susceptibility agar disc diffusion testing.

Multiple antibiotic resistance evaluation

Multiple antibiotic resistance (MAR) values for each isolate were calculated by summing the number of antibiotics to which the isolate was resistant and dividing by the total number of antibiotics assayed (Kaspar et al., 1990). The MAR values for each sample were calculated by summing the MAR values of all individual isolates and dividing by the total number of isolates per sample.

Statistical analysis

Changes in the number of birds carrying indicator organisms resistant to specific antibiotics over sampling dates were evaluated by using the G-log likelihood ratio; individual birds were the sampling unit. Changes in the proportion of individual isolates resistant to specific antibiotics were evaluated by using the two-tailed Z test; the total number of organisms resistant to a given antibiotic (fall, winter, spring) were the sampling units. The two-tailed t-test for two samples with unequal variances was used to evaluate differences between MAR values (Daniel, 1998). Statistical tests were performed by using SPSS software (SPSS, Base 10, Chicago, Illinois, USA).

	RESULTS

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The thermotolerant fecal indictor organism concentration for each sample was determined (Table 1). Enterococci were isolated from all 63 samples (fall, n = 21; winter, n = 25; spring, n = 17). The mean enterococcal concentration was 7.3x10⁵ CFU/0.1g (g^–1) wet weight of feces, with counts ranging from 1x10²–1x10⁷ CFU g^–1 wet weight of feces. E. coli were isolated from 47 samples (fall, n = 21; winter, n = 14; spring, n = 12). The mean E. coli concentration was 3.6x10⁵ CFU g^–1 wet weight of feces, with counts ranging from 0–1x10⁷ CFU g^–1 wet weight of feces. Of the 63 initial culture attempts, E. coli was not isolated from one fall (5%), 11 winter (44%), and seven spring (41%) samples. After overnight resuscitation in phosphate buffered saline, E. coli was isolated from the previously negative fall sample and from two each of the initially negative winter and spring samples.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Percentage of individual Enterococcus spp. and Escherichia coli isolates from migratory Canada geese resistant to 10 antibiotics, 2002–3. TET = tetracycline; CLT = chlortetracycline; PENG = penicillin G; AMP = ampicillin; CEPH = cephalothin; STR = streptomycin; GEN = gentamycin; CIP = ciprofloxacin; SUL = sulfathiazole; CHL = chloramphenicol.

The most common MAR pattern shown by E. coli was multiple resistance to chlortetracycline, penicillin G, ampicillin, cephalothin, and sulfathiazole (55.8% of total E. coli isolates). An additional 16.9% of total isolates were resistant to the above combination minus chlortetracycline. Only 0.02% of total E. coli isolates were resistant to less than four antibiotics, whereas 0.77% of total isolates were simultaneously resistant to seven different antibiotics.

Enterococcal colonies may be classified as either pigmented (yellow) or nonpigmented (white). The recognized human nosocomial pathogens E. faecalis and E. faecium are both nonpigmented. We identified all nonpigmented isolates from all winter and spring samples to determine whether E. faecalis or E. faecium were present. Only nine of 24 winter samples contained nonpigmented enterococci, whereas all spring samples contained these organisms. Of nonpigmented winter isolates, 44% were identified as E. faecalis and 56% were E. faecium. In the spring sample, 13% of nonpigmented isolates were E. faecalis and 31% were E. faecium. Other nonpigmented spring isolates were identified as E. durans and E. hirae. The fall samples were not evaluated for enterococcal species identity.

	DISCUSSION

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The mean concentration of E. coli in our sample (47 geese) was 6.6x10⁵ CFU g^–1 wet weight of feces, with seasonal averages ranging from 7.0x10⁴–1.3x10⁶ CFU g^–1 wet weight of feces. Of 236 geese sampled in Westchester, New York, USA, in 1995–97, the mean fecal coliform (FC) level was 1.53x10⁴ FC g^–1 wet weight of feces, with seasonal averages ranging from 5.2x10⁴–1.2x10⁷ FC g^–1 wet weight of feces (Alderisio and DeLuca, 1999). Total FC counts are expected to exceed E. coli counts because FC counts include Klebsiella and Enterobacter species as well as E. coli.

Successful recovery of E. coli from goose feces varied with the sampling date. Kullas et al. (2002) isolated fecal E. coli from 46% of October samples, 8% of January samples, and 25% of March samples from Canada geese in Fort Collins, Colorado, USA. They suggested a direct correlation between the mean ambient temperature and the recovery of E. coli, and they reported mean temperatures of 10.7 C, –0.5 C, and 8.9 C for sampling dates. Our recovery rates for E. coli for the same respective sampling months were 100, 56, and 70%. Our respective sampling temperatures were 8.8 C, 0.3 C, and 8.8 C. Differences in recovery rates between studies may reflect variation in sample collection and culture techniques or differences in the fecal flora of the sampled populations, or the differences may indicate greater sensitivity of some strains of E. coli to low temperatures. Brittingham et al. (1988) postulated that the isolation rate of E. coli from waterfowl may vary according to the exposure of the birds to fecal matter from other vertebrate species. Fallacara et al. (2001) suggested waterfowl might acquire E. coli and other pathogens from water sources contaminated with human sewage or agricultural runoff. It is possible that in winter months when many surface water sources are frozen, migratory waterfowl may have limited access to environmental E. coli sources, thus decreasing the recovery rates during the coldest months.

The MAR value calculated for a given organism or sample depends on the specific panel of antibiotics chosen for the profile. As such, MAR values are primarily useful for comparing the resistance patterns of bacterial strains within a sample and for determining the range of antibiotic resistance determinants present within a sample population. We found a wide range of MAR patterns in both the enterococci and E. coli isolates of migratory birds, indicating great diversity in the microbial flora of these birds. The lower number of MAR patterns observed in the spring E. coli samples may be a reflection of a reduced sample size (12 birds).

MAR profiles have been used to differentiate point source from nonpoint source E. coli populations (Kaspar et al., 1990; Parveen et al., 1997). By analyzing MAR patterns using discriminant analysis, microbial source tracking has allowed the identification of the source of fecal pollution of surface waters (Wiggins, 1996; Harwood et al., 2000). However, E. coli isolates from goose feces were examined and found to be very poorly classified by microbial source tracking (0% correct classification) (Guan et al., 2002).

Enterococci have intrinsic resistance to the cephalosporins, often have high level resistance to aminoglycosides (streptomycin and gentamycin), and are developing widespread resistance to penicillin and ampicillin (Jeljaszewicz et al., 2000). All sampled geese harbored enterococci that were resistant to streptomycin and sulfathiazole, and more than 95% of geese carried enterococci resistant to cephalothin and gentamycin. The differences in the MAR profiles of the enterococci may, in part, reflect that several species comprise the enterococcal flora of migratory geese, with each species having different antibiotic resistance patterns.

Although the enterococci are pathogenic only under specific conditions, they are now among the most common causes of human nosocomial infections (Jeljaszewicz et al., 2000). The nonpigmented enterococci, E. faecalis and E. faecium, are the species most commonly associated with clinical infection. Enterococcus faecalis is considered the more pathogenic species because it is more likely to carry human virulence factors (Eaton and Gasson, 2001). Wheeler et al. (2002) proposed using E. faecalis as a human fecal indicator for microbial source tracking. They found that E. faecalis was present only in humans, dogs, and chickens. They examined three Canada geese and isolated E. faecium but not E. faecalis. We examined the feces of 40 birds and found that 15% of the geese carried E. faecalis, and 25% carried E. faecium. Geese defecate freely in ponds and coastal waters within their migration paths. Perhaps the suggestion that E. faecalis be used as an indicator of human fecal pollution may need to be reassessed for areas frequented by migratory Canada geese.

	ACKNOWLEDGMENTS

 
We thank Dr. Grace Wyngaard for assistance with statistical analyses. We thank Dr. Kate Verville and Dr. Paul Bologna for critical reading and helpful comments. We also thank Fairleigh Dickinson University for a faculty Grant in Aid.

	LITERATURE CITED

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Received for publication 23 October 2003.

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