Abundance and characteristics of the recreational water quality ...
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Abundance and characteristics of the recreational waterquality indicator bacteria Escherichia coli and enterococciin gull faeces
L.R. Fogarty1, S.K. Haack1, M.J. Wolcott2 and R.L. Whitman3
1US Geological Survey, Lansing, MI, 2US Geological Survey, Madison, WI, and 3US Geological Survey, Porter, IN, USA
2002/326: received 3 September 2002, revised 8 January 2003 and accepted 23 January 2003
ABSTRACT
L.R . FOGARTY, S.K . HAACK, M.J . WOLCOTT AND R.L . WHITMAN. 2003.
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� 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 (105–109 CFU g)1) and enterococci (104–
108 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.
Keywords: enterococci, Escherichia coli, faecal contamination, gulls, water quality.
INTRODUCTION
Recreational waters are susceptible to a variety of sources of
microbiological pollution (USEPA 1986; Levesque et al.
2000; Rose et al. 2001). In 1986, the United States
Environmental Protection Agency (USEPA) published
numerical standards for Escherichia coli and enterococci for
fresh US recreational waters (USEPA 1986). In October
2000, the US Congress required states with coastal (marine
or Great Lakes) recreational waters to adopt (by April 2004)
the USEPA criteria, and to establish monitoring and
public notification programmes. As the responsible agencies
develop beach-monitoring programmes in response to the
new legislation, new and more detailed informationCorrespondence to: L.R. Fogarty, USGS-WRD, 6520 Mercantile Way Suite 5,
Lansing, MI 48911, USA (e-mail: [email protected]).
ª 2003 The Society for Applied Microbiology
Journal of Applied Microbiology 2003, 94, 865–878
concerning sources of E. coli and enterococci will be
required to manage Great Lakes recreational waters.
Because faecal contamination can come from many different
human (septic systems or sewers) and animal sources
(animal pasture runoff, waterfowl, wildlife or domestic
animals), identifying sources of faecal contamination can be
an aid to improving management of recreational waters.
Recently, various methods have been proposed to identify
sources of faecal contamination by classifying faecal bacteria
[faecal coliforms, faecal streptococci (more recently, entero-
cocci) or E. coli] from known sources based on phenotypic or
genotypic characteristics and using these characteristics to
classify faecal bacteria of unknown source that were isolated
from the environment. Phenotypic source-determination
methods have included multiple antibiotic resistance (MAR)
profiles (Krumperman 1983; Kaspar et al. 1990; Wiggins
1996; Parveen et al. 1997; Hagedorn et al. 1999; Harwood
et al. 2000), O-serotyping and fatty acid methyl ester
analysis (Parveen et al. 2001). Genotypic source-determin-
ation methods have included ribotyping (RT; Parveen et al.
1999; Carson et al. 2001), pulsed-field gel electrophoresis
(Parveen et al. 2001) and rep-PCR profiles (Dombek et al.
2000).
Very seldom has the range of phenotypic or genotypic
characteristics of faecal indicator bacteria within host
populations been considered in source-determination
studies. Even though early studies indicated that individ-
ual animals host a variety of phenotypes and genotypes of
E. coli including resident strains and continuous immi-
grants from the environment (Selander et al. 1987), most
source-determination studies have used samples taken on
multiple dates or from multiple locations. For example,
Wiggins et al. (1999) studied antibiotic resistance profiles
of faecal streptococci isolated from humans, cattle, poultry
and wild animals over a 4-year period, and classified the
isolates with respect to source, using discriminant analysis.
The average rate of correct classification (ARCC; number
of correctly classified isolates divided by the total number
studied) was 64–78%. It was hypothesized in this study
that the relatively low ARCC might have been because of
changes within the source-specific populations from which
the samples were collected. Other studies applying
discriminant analysis to different source-tracking methods
have reported ARCCs of: RT, 82% (Parveen et al. 1999)
or 74–96% depending on number of sources analysed
(Carson et al. 2001); rep-PCR, 87–93% (Dombek et al.
2000); MAR of faecal streptococci, 87% (Hagedorn et al.
1999); MAR of faecal streptococci or faecal coliforms,
62–64% (Harwood et al. 2000). Additionally, most source-
determination studies have shown varying success of
classification depending on source type. For example,
Dombek et al. (2000) used rep-PCR DNA fingerprints as
a method of source determination for E. coli isolated from
faecal samples. In that study, 100% of the E. coli isolates
from chickens and cows were classified correctly; however,
only 80–89% of the E. coli isolates from waterfowl (ducks
and geese) were classified correctly. Evaluation of variab-
ility in the physiological and genomic characteristics of
faecal bacteria populations within hosts may help to
explain the percentage of incorrect classification of source
samples and refine the usefulness of the proposed source-
determination methods.
Gulls (Larus sp.) have not been addressed in any
source-determination studies to date, despite their poten-
tial or documented significance as a major source of faecal
contamination to reservoirs and recreational waters and at
bathing beaches (Jones et al. 1978; Levesque et al. 1993,
2000; Hatch 1996; Alderisio and Deluca 1999; Jones and
Obiri-Danso 1999; Obiri-Danso and Jones 2000). Gull
faecal material is considered a threat to human health
(Hatch 1996; Levesque et al. 2000). Studies have docu-
mented the presence in gull faeces of human bacterial
pathogens such as Salmonella spp., Aeromonas spp.,
Campylobacter spp, and E. coli serotype O157 (Jones et al.
1978; Hatch 1996; Wallace et al. 1997; Levesque et al.
2000; Obiri-Danso and Jones 2000). No studies have
specifically reported the numbers of USEPA-recommen-
ded recreational water faecal indicator bacteria E. coli and
enterococci in gull faeces, although faecal coliforms
(Levesque et al. 1993, 2000; Alderisio and Deluca 1999)
and faecal streptococci (Jones and Obiri-Danso 1999) have
been reported. Information on the abundance and phen-
otypic and genotypic characteristics of these indicator
bacteria in gull faeces will be useful to managers of
recreational waters in the US Great Lakes and similar
environmental settings.
The objective of this study was to evaluate, over the
typical recreational swimming season, numbers and selected
phenotypic and genotypic characteristics of the indicator
bacteria E. coli and enterococci in gull faeces. We quantified
E. coli and enterococci in gull faeces collected at two Lake
Michigan, USA, beaches between May and October 2000.
We used E. coli rep-PCR genomic profiles and E. coli and
enterococci phenotypic tests to characterize E. coli and
Enterococcus populations in gull faecal material. This paper
presents the results of this study.
MATERIALS AND METHODS
Gull faecal sample collection
Gull faecal samples were collected at Lake Michigan
beaches in Chicago, IL, USA (CHI) and Traverse City,
MI, USA (TC) between May and October 2000. These
beaches lie on the opposite shores of Lake Michigan
(Figure 1). Sample nomenclature is presented in Table 1.
866 L.R. FOGARTY ET AL.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
Faecal samples were collected just after defecation. CHI
gull faecal samples were collected by rolling a sterile swab
in the centre of gull faecal droppings. TC samples were
collected with a sterile spatula. Care was taken during
sampling to be sure no surrounding beach sediment was
collected. Samples were placed in a sterile tube, stored on
ice, and processed in the laboratory 24–48 h after sample
collection. Faecal material weight was determined for CHI-
A21 and all TC samples but not for CHI-JU or CHI-A1
samples. All samples were suspended in a known volume
of phosphate buffered saline, diluted in series and filtered
by membrane filtration method for isolation of E. coli and
enterococci.
Bacteria isolation and identification
Escherichia coli and enterococci were isolated from all
samples using membrane filtration (American Public Health
Association 1998; USEPA 2000). A sterile buffered saline
control and a series of dilutions were passed through
individual sterile 0Æ45 lm pore size, gridded cellulose nitrate
membrane filters (Advantec MFS, Inc., Pleasanton, CA,
USA). Dilution tubes were thoroughly mixed before
filtration. Total coliform bacteria were identified on
mENDO agar LES medium (DIFCO Laboratories, Detroit,
MI, USA). For E. coli identification, membranes with
15–50 well-separated coliform colonies were transferred to
300 km
lllionois
Michigan
United States
Canada
Chicago
TraverseCity
0
85˚ 45′
45˚ 00′
Lake
Mic
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n
Fig. 1 Study location
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ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
Nutrient Agar containing 4-methylumbelliferyl-b-DD-glu-
curonide (Na-MUG agar; DIFCO). Colonies that fluoresced
blue under UV light were identified presumptively as E. coli.
Selected presumptive E. coli were transferred to LB medium
and confirmed by three different physiological tests: negat-
ive indophenol oxidase production (BBL� oxidase reagent
droppers, Becton Dickinson Microbiology Systems, Coc-
keysville, MD, USA), positive b-DD-galactosidase reaction
(Taxo� ONPG discs, Becton Dickinson Microbiology
Systems), and positive indole production (BBLTM Dry-
SlideTM Indole, Becton Dickinson Microbiology Systems).
CHI isolates subsequently were verified as E. coli based on
reactions from the Vitek� GNI+ system (bioMerieux,
Hazelwood, MO, USA).
Enterococcus isolates were identified using membrane
filtration on mEI agar as described by USEPA (USEPA
2000). Enterococci with representative morphologies on
mEI agar were isolated and confirmed by growth on brain
heart infusion agar with 6Æ5% NaCl at 35�C, esculin
hydrolosis on bile esculin agar and negative catalase activity
(USEPA 2000). Enterococcus isolates were further charac-
terized using multiple physiologic assays (API� rapid ID 32
Strep, bioMerieux) as well as colony colour and haemolysis
on Columbia sheep blood agar (BBL Becton Dickinson).
Rep-PCR genomic profiles of Escherichia coliisolates
Rep-PCR procedures were revised slightly from those
described in Rademaker and de Bruijn (1997). Primers used
were REP 1R and REP 2I (Versalovic et al. 1991) obtained
from Genosys Biotechnologies (The Woodlands, TX, USA)
and diluted in TE (10 mmol l)1 Tris, pH 8Æ0, 1 mmol l)1
EDTA). The rep-PCR reaction components consisted
Table 1 Numbers of Escherichia coli, E. coli genotypes and enterococci in gull faecal samples
E. coli Enterococci
Seagull faecal sample Collection date CFU No. of isolates No. of genotypes* CFU No. of isolates
CHI-Ju-A 26 June 2000 – 3 2 – 5
CHI-Ju-B 26 June 2000 – 3 3 – 5
CHI-Ju-C 26 June 2000 – 8 7 – 5
CHI-Ju-D 26 June 2000 – 6 6 – –
CHI-Ju-E 26 June 2000 – – – – 5
CHI-Ju-F 26 June 2000 – – – – 5
CHI-A1-B5 1 August 2000 – 2 2 – –
CHI-A1-B6 1 August 2000 – 3 3 – –
CHI-A1-B7 1 August 2000 – 2 2 – –
CHI-A1-B8 1 August 2000 – 4 4 – –
CHI-A1-SB1 1 August 2000 – 1 1 – –
CHI-A1-SB2 1 August 2000 – 3 3 – –
CHI-A1-SB3 1 August 2000 – 2 2 – –
CHI-A1-SB4 1 August 2000 – 2 2 – –
CHI-A21-A 21 August 2000 1Æ9 · 109 10 9 4Æ0 · 106 5
CHI-A21-B 21 August 2000 2Æ3 · 107 10 7 2Æ8 · 105 6
CHI-A21-C 21 August 2000 1Æ9 · 107 5 5 6Æ5 · 107 5
CHI-A21-D 21 August 2000 5Æ0 · 106 9 3 2Æ0 · 104 10
TC-My-1 24 May 2000 5Æ7 · 106 2 2 – –
TC-My-2 25 May 2000 1Æ6 · 106 10 8 2Æ1 · 106 –
TC-A-A 9 August 2000 <1 · 105� – – 4Æ0 · 106 –
TC-A-B 9 August 2000 1Æ8 · 107 10 8 1Æ3 · 108 5
TC-A-C 9 August 2000 <1 · 106� – – <1 · 106� –
TC-A-D 9 August 2000 1Æ4 · 107 – – 2Æ4 · 108 5
TC-Oc-A 14 October 2000 6Æ9 · 107 4 3 2Æ3 · 107 1
TC-Oc-B 14 October 2000 1Æ0 · 106 3 2 1Æ0 · 106 1
Total 102 84 63
*Different genotypes defined as isolates having rep-PCR profiles of <100% similarity.
�Number less than the lowest dilution tested, as indicated.
CFU, colony-forming unit; CHI, Chicago; TC, Traverse City.
868 L.R. FOGARTY ET AL.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
of a final concentration of: 1 · PCR reaction buffer
(100 mmol l)1 Tris–HCl pH 8Æ5, 500 mmol l)1 KCl; Gibco
BRL, Gaithersburg, NY, USA), 3Æ3 mmol l)1 MgCl2,
125 lmol l)1 of each dNTP (Pharmacia, Piscataway, NJ,
USA), 0Æ01 lg ll)1 BSA (Boehringer Mannheim, Indianap-
olis, IN, USA), 10% DMSO, 2 lmol l)1 of each primer, 2U
Taq DNA Polymerase (Gibco BRL), 1 ll of a 1 : 10 diluted
E. coli culture (18–24 h culture in LB broth), and sterile
tissue culture water to bring the volume up to 25 ll. To
confirm purity, cultures used for the PCR were streaked
onto EMB (DIFCO) and TSA with 5% sheep blood (BBL
Becton Dickinson). DNA amplification was carried out in a
Perkin Elmer 2400 Gene Amp PCR system (Perkin Elmer-
Cetus, Norwalk, CN, USA) with the following conditions:
95�C for 7 min; 34 cycles of: 94�C for 3 s, 92�C for 30 s,
40�C for 1 min, 65�C for 8 min; a final elongation of 16 min
at 65�C; and a final hold at 4�C. PCR products (7 ll of each)
were electrophoresed on a 2% agarose gel for 100 min at
75 V in a Wide Mini-Sub Cell GT system (Bio-Rad
Laboratories, Hercules, CA, USA) and visualized with
ethidium bromide staining. On each gel, a laboratory strain
of E. coli (ATCC 25922) was included as a positive control
and standard for comparisons. Banding patterns of scanned
images were compared using BioNumerics version 2Æ5(Applied Maths, Kortrijk, Belgium), with a resolution of
600 dpi. Similarities between banding patterns were estab-
lished using unweighted pair-group method using arith-
metic averages (UPGMA) clustering, based on the Dice
correlation coefficient with 1Æ0% optimization, 2% position
tolerance and 2% minimum height.
Enterococci biochemical tests
Enterococcus faecalis ATCC 19433 was used as control for
each series of tests. Results were interpreted according to
API� rapid ID 32 Strep manufacturer’s instructions. API�
rapid ID 32 Strep tests only indicate eight Enterococcus
species: Ent. avium, Ent. casseliflavus, Ent. durans, Ent.
faecalis, Ent. faecium 1 or 2, Ent. gallinarum, Ent. hirae and
Ent. saccharolyticus. For the purpose of discussion, species
names were assigned to isolates based on the API� test
results, although it is broadly recognized that physiologic
tests are inadequate to definitively identify enterococci from
environmental sources (Muller et al. 2001; Svec et al. 2002).
To establish similarities between isolates, the API� test
responses for the Ent. faecalis ATCC control, all gull
Enterococcus isolates and the eight Enterococcus species
reported in the API� manual were converted to binary data
and cluster analysis of the binary response profiles was
conducted (agglomerative clustering, Ward method) using
S-Plus 2000 (MathSoft Inc., Seattle, WA, USA). Resistance
of enterococci to the antibiotics streptomycin, gentamicin,
tetracycline, vancomycin and ampicillin was determined
using the Etest� (AB Biodisk, Piscataway, NJ, USA). These
five antibiotics are commonly used to treat enterococcal
infections in humans, and levels of resistance to each
antibiotic were defined using standard criteria (National
Committee for Clinical Laboratory Standards 2002).
RESULTS
Abundance of Escherichia coli and enterococciin gull faeces
Escherichia coli concentrations ranged from <1Æ0 · 105–
109 g)1 of faeces, and enterococci ranged from 104 to 108 g)1
(Table 1). The mean number (± standard deviation) of E. coli
for Chicago faecal samples was 4Æ9 · 108 ± 9Æ4 · 108 g)1
and for Traverse City faecal samples it was 1Æ4 · 107 ±
2Æ3 · 107 g)1. For Chicago faecal samples, enterococci
numbers ranged from 104 to 107 CFU g)1 (mean: 1Æ7 ·107 ± 3Æ2 · 107 g)1) and for Traverse City samples entero-
cocci numbers ranged from 105 to 108 (mean: 5Æ7 · 107 ±
9Æ3 · 107 g)1).
Genotypic and phenotypic characteristics ofEscherichia coli from gull faeces
Chicago isolates. Escherichia coli isolates from Chicago
seagull samples were characterized by both rep-PCR
genomic profiles and Vitek� phenotype. Clustering of these
isolates based on their rep-PCR profiles, and the relation of
these profiles to Vitek� phenotype, is depicted in Figure 2.
Isolates having <100% similarity of banding patterns
(Figure 2) were defined as different genotypes. In every
case, CHI isolates with identical genotypes were obtained
only from single seagull faecal samples (Figure 2). Typically,
isolates with identical genotypes also had identical or very
similar phenotypes. There was no unique phenotype for
each rep-PCR banding pattern at the 100% similarity level,
primarily because the isolates exhibited variability in only 12
phenotype characters, resulting in less phenotypic variation
than genotypic variation. At approximately 85% similarity
of banding patterns, clusters tended to be characterized by
isolates from a single sampling date (although not necessarily
the same sample). Clusters at approximately 85% banding
pattern similarity also tended to share phenotypic features,
and tended to be distinct in phenotype from adjacent
clusters at lower levels of similarity. There was some
association between phenotype and sampling date. The only
isolates positive for all phenotypic tests were from the A21
sampling event (faecal samples A21 B and C). Failure to
utilize raffinose was more common in June than August
samples. At >70% similarity, seven clusters (A–G in
Figure 2) with broadly similar banding pattern and phen-
otypic features could be identified.
E. COLI AND ENTEROCOCCI IN GULL FAECES 869
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
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ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
Traverse City isolates. Traverse City isolates were char-
acterized only by rep-PCR genomic profiles. When rep-PCR
profiles for TC isolates were analysed with those for the
CHI isolates (Figure 3), seven of 29 TC isolates fell into the
existing CHI clusters A–D, some as much as 90% similar to
CHI isolates, and relations among Chicago isolates in these
clusters remained essentially unchanged (Figure 3). The
remaining 22 TC isolates formed new, TC-only, groups at
£70% similarity with CHI isolates in clusters D–G. As for
the CHI isolates, identical TC genotypes were only
identified in single gull faecal samples (Figure 3). There
was no case in which the same genotype was found in both
CHI and TC gull faecal samples.
Number of genotypes per sample. No prior information
was available on gull E. coli intraspecies population genomic
variability; therefore, we collected multiple isolates from all
but one gull faecal sample (Table 1). The number of
different E. coli genotypes was determined for each gull
faecal sample (Table 1). Every faecal sample with more than
one isolate yielded multiple E. coli genotypes (Table 1).
There was a tendency for the number of genotypes to
increase with increasing number of isolates per sample, with
a median of eight genotypes for the four samples with 10
isolates (Figure 4).
Phenotypic characteristics of enterococcifrom gull faeces
API test responses. Enterococcus phenotypes differed
between sites and on different sampling dates within sites.
The results of cluster analysis of API� Rapid ID 32 Strep
test responses for 51 CHI enterococci and 12 TC enterococci
are shown in Figure 5. Cluster A is composed of enterococci
exhibiting similar API� test responses to those of Ent.faecalis or Ent. avium. This cluster was dominated by 23 of
the 25 June isolates from CHI gull faecal samples. In
addition, TC isolates from October fell in this cluster.
Cluster B was composed of enterococci having similar test
responses to those of Ent. gallinarum, Ent. durans, Ent. hirae
and Ent. faecium. The species Ent. durans, Ent. hirae and
Ent. faecium form a highly related phylogenetic group, and
are difficult to distinguish based on biochemical tests
(Devriese et al. 2002). The two remaining isolates from
June CHI samples fell in Cluster B, along with the majority
of August TC isolates. Finally, Cluster C was dominated by
22 of the 26 August CHI isolates and included two TC
August isolates. The majority of the isolates in Cluster C
could not be identified by the API� Rapid ID 32 Strep test.
Group C1 was composed of enterococci uniformly positive
for mannitol, sorbitol and ribose acidification, positive for
arginine dihydrolase and alkaline phosphatase, and variably
positive for LL-arabinose acidification. The majority of Group
C2 isolates exhibited yellow pigmentation. API� Rapid ID
32 Strep only identifies the yellow-pigmented Ent. casse-
liflavus. Only the two TC August isolates exhibited the
correct biochemical test responses to be classified as Ent.
casseliflavus by the API� tests. The remaining isolates in
Group C2 were all isolates from CHI August gull faecal
samples.
Antibiotic resistance. Enterococci from CHI and TC
exhibited a variety of resistance patterns to the five tested
antibiotics (Table 2) with no obvious pattern with respect to
sampling date or location. The highest level of streptomy-
cin tested was 256 lg ml)1, and several isolates were fully
resistant at this level. It is possible that some of these
isolates may be resistant at levels >1000 lg ml)1, which
would indicate acquired resistance. Several isolates were
resistant to tetracycline at the highest tested concentration
(256 lg ml)1). One isolate was resistant to the highest
concentration of gentamicin tested, and at a level indicating
acquired resistance. No isolate was resistant to ampicillin.
No isolate exhibited resistance to vancomycin; however,
susceptibility to this antibiotic is defined as £4 lg ml)1. The
three isolates exhibiting an inhibitory concentration of
6 lg ml)1 were all yellow-pigmented isolates (two identified
as Ent. casseliflavus) and intrinsic, intermediate levels of
vancomycin resistance are typical of some yellow-pigmented
enterococci, including Ent. flavescens and Ent. casseliflavus.
DISCUSSION
The high concentrations of both the E. coli and the
enterococci associated with gull faeces suggest that gulls
Fig. 2 Dendrogram (UPGMA clustering based on Dice correlation
coefficient) of 73 Escherichia coli rep-PCR profiles for isolates obtained
from gull faeces collected on beaches in Chicago, IL. Phenotypes based
on Vitek� GNI+ response are shown but were not used in the
clustering; j ¼ a positive response, u ¼ a negative response. [DP-3
(DP-300 fermentation), OFG (glucose, oxidative utilization), GC
(growth control), ACE (acetamide utilization), ESC (esculin hydroly-
sis), PLI (Plantindican reaction), URE (urea utilization), CIT (citrate
utilization), MAL (malonate utilization), TDA (tryptophan deami-
nase), PXB (Polymixin B growth), LAC (lactose oxidation), MLT
(maltose oxidation), MAN (mannitol oxidation), XYL (xylose oxida-
tion), RAF (raffinose utilization), SOR (sorbitol utilization), SUC
(sucrose utilization), INO (inositol utilization), ADO (adonitol util-
ization), COU (p-coumaric fermentation), H2S (hydrogen sulphide
production), ONP (ortho-nitrophenol galactopyranoside hydrolysis),
RHA (rhamnose utilization), ARA (LL-arabinose utilization), GLU
(glucose fermentation), ARG (argenine dihydrolation), LYS (lysine
decarboxylation), NC (decarboxylation control), ORN (ornithine
decarboxylation)]. Isolate designations: CHI – Chicago; JU – June, A1
– early August, A21 – late August
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E. COLI AND ENTEROCOCCI IN GULL FAECES 871
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
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A21A21A21A21OCA21A21A21A1A1A21A1A21JUA21JUJUA1MYA1MYA1A1A1A1A1MYA21A21A21JUMYJUA21JUJUJUA1A1A21A1MYA21A21A21A21A21A21A21A21A21AA1A21OCOCAOCMYMYMYJUJUJUJUJUJUAAAAA21A21A21A21A1JUA21AAAA21JUJUMYMYJUA1A1MYJUJUA1A21A21AOCOCOCA1A21MY
AAAAAABCSB2B8AB8CBBDCB82SB32SB2SB2SB1B8SB42BBCB1DCAAAB7B5AB71DDDDDDDDDBB6BAABA222CCCCCDBBBBBBBBB6DCBBBACC22BSB4B62DDSB3AABBBBB5B2
6845B2511271832613A3J3
241K4351C423211292B3121049856K26ECHALHG547867BDCA97810414FJG132FC42B1E522310ECBD11D
Similarity (%)
Dat
e
Sam
ple
Isol
ate
A
B
C
D
G
E
E
F
872 L.R. FOGARTY ET AL.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
may be a significant source of these indicator bacteria
recommended by the USEPA for monitoring recreational
waters (USEPA 2000). Other authors have noted the large
numbers of faecal bacteria associated with the faeces of gulls
and other shore birds and have documented the impact gull
faeces may have on bacterial contamination of reservoirs,
beach sediments and coastal waters (Jones et al. 1978;
Levesque et al. 1993, 2000; Hatch 1996; Alderisio and
Deluca 1999; Jones and Obiri-Danso 1999; Obiri-Danso and
Jones 2000). However, no prior studies have specifically
addressed the number of E. coli or enterococci in gull faeces.
Levesque et al. (1993, 2000) reported that 95–99% of faecal
coliforms in gull faeces were E. coli. E. coli concentrations
per gram of gull faeces in our study (<1Æ0 · 105 to 1Æ9 · 109,
Table 1) were similar to faecal coliform concentrations
found in other studies (1Æ1 · 106 to 1Æ1 · 1010; Levesque
et al. 1993; Alderisio and Deluca 1999; Jones and Obiri-
Danso 1999). A similar range in the concentration of
enterococci per gram of faeces (2Æ0 · 104 to 2Æ4 · 108) was
also observed in our study. Gould and Fletcher (1978)
determined that the average wet weight of faeces excreted by
different gull species ranged from 11Æ2 to 24Æ9 g day)1. This
would result in an average daily load of E. coli and entero-
cocci from one gull on the Chicago beach up to 1Æ2 · 1010
and 4Æ2 · 108, respectively (3Æ5 · 108 and 1Æ4 · 109,
respectively, for Traverse City).
We observed no temporal or geographic trend in E. coli or
enterococci concentration in gull faeces. Likewise, Levesque
et al. (2000) demonstrated little difference in the numbers of
faecal coliforms in gull faecal material with respect to gull
age group, colony or sampling date. In addition, a study by
Alderisio and Deluca (1999) indicated a fairly stable
concentration of faecal coliforms (107–108 g)1) over four
seasons across two sampling years.
Within the Great Lakes region, populations of the ring-
billed gull (Larus delawarensis) have been increasing in
recent years, especially in Illinois (Sauer et al. 2002). In
recent years, more than 7000 breeding pairs of ring-billed
gulls were counted at six sites along the Chicago shoreline,
and as many as 17 700 breeding pairs at nearby locations
outside the Chicago metropolitan area (F. Cuthbert, Uni-
versity of Minnesota, Minneapolis, MN, USA, personal
communication). As many as 13 000 breeding pairs have
been counted at an island located west of Grand Traverse
Bay. There are over 250 000 breeding pairs of ring-billed
gulls in the Great Lakes region, accompanied each year by
non-breeding immatures up to 2 years of age. Other water
birds found along the Chicago and Traverse City shorelines
include mallard ducks and Canada geese, for which the
population numbers have also been increasing in the Great
Lakes region (Sauer et al. 2002). However, Canada geese
were rarely seen on the beaches during the swimming
season, and mallard ducks were less numerous than gulls at
both beaches.
As noted by others for both Europe and the US (Hatch
1996; Jones and Obiri-Danso 1999), large and increasing
populations of ring-billed gulls may bring increased risk of
human exposure to endemic bacterial pathogens (Campylo-
bacter spp.) as well as those acquired through feeding at
landfills, animal pastures and sewage disposal sites (Salmon-
ella, other enteric bacteria). The increasing populations of
ring-billed gulls in the Great Lakes, combined with the large
numbers of faecal bacteria they carry, may constitute a major
non-point source of water pollution. With this concern in
mind, means to discriminate gull faecal pollution from other
potential sources would be especially valuable to beach
managers and water pollution control authorities.
Our results indicate a high degree of intra-species
population variation for E. coli (defined by rep-PCR profiles
and Vitek� biotype) in gull faeces taken from Lake
Michigan beaches. Rep-PCR has been shown to be a
powerful method for identifying intra- and inter-species
genotypic relations, and rep-PCR profiles have been shown
to be correlated with intra-specific phenotypic characteris-
tics in Xanthomonas, Pseudomonas and Ochrobactrum spp.
(Lemanceau et al. 1995; Lebuhn et al. 2000; Rademaker
et al. 2000). Johnson and O’Bryan (2000) showed that E. coli
rep-PCR profiles were related to E. coli clusters based on
multiple locus enzyme electrophoresis (MLEE). In our
study, E. coli isolates with identical genotypes had very
similar or identical Vitek� phenotypes. Although our sample
set was small, we found a median of eight genotypes in four
Fig. 3 Dendrogram (UPGMA cluster analyses based on Dice corre-
lation coefficient) of Escherichia coli rep-PCR profiles for 73 isolates
obtained from gull faeces collected on beaches in Chicago, IL (CHI)
and 29 isolates from gull faeces collected at Traverse City, MI (TC)
beaches. Isolate designations as for Figure 2, except TC – Traverse
City; A – August; OC – October
y = 3·1861Ln(x) – 0·3422
R2 = 0·7852
0123456789
10
0 2 4 6 8 10 12
Number of isolates
Num
ber
of g
enot
ypes
Fig. 4 Relation between number of Escherichia coli rep-PCR geno-
types and number of isolates obtained per gull faecal sample
b
E. COLI AND ENTEROCOCCI IN GULL FAECES 873
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
casseliflavus
durans
A
CHI A21 C1
CHI A21 C5
Distance
TC A D4 casseliflavusTC A D3 casseliflavus
CHI A21 B2BCHI A21 B4
CHI A21 C4CHI A21 A6CHI A21 A2
CHI A21 A5CHI A21 A3
CHI A21 D1CHI A21 D4CHI A21 B5CHI A21 B6
CHI A21 A1
CHI A21 D2CHI A21 D3
CHI A21 D5
CHI A21 D6CHI A21 D8
CHI A21 D9CHI A21 D7
CHI A21 D10hiraegallinarum
TC A D5 hirae
TC A B2TC A D2 durans
TC A D1hirae
TC A B1 duransTC A B5TC A B4 durans
TC A B3 durans faecium 1faecium 2CHI JU C3 faecium 1CHI JU C1gallinarumCHI JU C8 faecalis
CHI A21 B1CHI JU E1 faecalisCHI JU C2
CHI A21 C3saccharolyticus
CHI JU E4 faecalisCHI JU E3 faecalis
CHI JU E2 faecalis
CHI A21 C2CHI JU C6
CHI JU F6 faecalis
CHI JU B3 faecalis
CHI JU B5CHI JU F2 faecalis
CHI JU E5 faecalis
CHI JU F5
CHI JU F3
CHI JU B2 faecalis
CHI JU A5 faecalis
CHI JU A1 aviumCHI JU B1 avium
avium
CHI JU B4CHI JU F4CHI JU A4
CHI A21 B2A
CHI JU A3TC OC B faecalis
TC OC A faecalisCHI JU A2
B
C
0·00·51·01·5
874 L.R. FOGARTY ET AL.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
faecal samples for which we collected 10 isolates, all but one
faecal sample with multiple isolates yielded more than one
genotype, and we found no instance where identical
genotypes occurred in more than one seagull faecal sample.
Our data suggest that large numbers of E. coli isolates from
gull faecal samples, and more than one isolate per sample,
would be required to fully characterize intra-specific pop-
ulation diversity, and to adequately characterize the popu-
lation for source-determination studies.
We observed structure in the rep-PCR profile clusters at
‡85% and around 70% similarity. CHI isolates that clustered
at ‡85% similarity were typically from the same sampling
date, shared identical or highly similar phenotypes, and
retained their close association even after TC isolates were
added to the data set. Five TC isolates were 85–90% similar
to CHI isolates. The close association of some CHI and TC
isolates in the same clusters suggests some population overlap
at the two geographically distinct sites. At >70% similarity of
banding patterns, CHI and TC isolates exhibited broadly
similar banding patterns, and for CHI isolates, such clusters
were accompanied by some unique phenotypic characteris-
tics (Figure 2). Previous studies (Lemanceau et al. 1995;
Johnson and O’Bryan 2000; Lebuhn et al. 2000; Rademaker
et al. 2000) have shown that large and consistent variations in
banding pattern have intra-specific genotypic and phenotypic
significance. Much more data on other phenotypic and
genotypic features of our isolates would be required to
establish the population significance of these broad clusters.
Nevertheless, our results suggest that several intra-species
groups of E. coli occur within gull faeces at two geograph-
ically separated Lake Michigan beaches and that the
proportion of isolates in these groups varies temporally.
Fig. 5 Dendrogram (agglomerative clustering, Ward method) of
binary API� Rapid ID 32 Strep biochemical response profiles of
enterococci isolates obtained from gull faeces collected on beaches in
Chicago, IL, and Traverse City, MI, compared to the standard
responses (shown in italics) of Ent. avium, casseliflavus, durans, faecalis,
faecium 1, faecium 2, gallinarum, hirae, and saccharolyticus. Isolate
designations: CHI, Chicago; TC, Traverse City; JU, June; A21 or AG,
August; OC, October
b
Table 2 Antibiotic resistance patterns of
enterococci from gull faecesAntibiotic resistance level (lg ml)1)
Isolate Cluster* Date Strep, >1000� Gent, >500� Van, 8–16� Tet, ‡16§ Amp, ‡16§
CHI JU B3 A1 JU >256 64 2 >256 1
CHI JU E5 A1 JU >256 64 2 >256 1
CHI JU F6 A1 JU >256 16 4 0Æ5 0Æ5CHI JU A2 A2 JU 128 16 1 0Æ125 0Æ19
CHI JU F4 A2 JU >256 24 4 1 0Æ5TC OC A A2 OC >256 64 3 64 0Æ75
TC OC B A2 OC >256 24 3 48 0Æ25
CHI JU B1 A3 JU 64 4 1Æ5 0Æ38 0Æ38
CHI JU C8 A4 JU >256 12 1 0Æ25 0Æ5CHI JU E4 A4 JU 48 4 1 0Æ38 0Æ25
CHI JU C3 B JU 32 12 0Æ75 48 1Æ5TC AG B2 B A >256 1024 3 >256 3
TC AG B3 B A >256 48 0Æ38 >256 2
TC AG D1 B A 96 24 2 0Æ75 0Æ38
CHI A21 B6 C1 A >256 16 2 0Æ38 0Æ5CHI A21 D2 C1 A 128 12 2 0Æ38 0Æ5CHI A21 D4 C1 A >256 24 2 128 0Æ5CHI A21 D9 C1 A >256 48 2 >256 0Æ75
CHI A21 A2 C2 A 16 8 6 0Æ75 0Æ5CHI A21 B4 C2 A 32 12 3 0Æ25 2
CHI A21 C5 C2 A 24 6 4 0Æ25 0Æ38
TC AG D3 C2 A 32 8 6 1 1
TC AG D4 C2 A >256 24 6 96 3
*Cluster as shown in Figure 5.
�Concentration above which isolate should be tested for high-level resistance.
�Range for intermediate resistance requiring further testing.
§Defined resistance level.
Strep, streptomycin; Gent, gentamicin; Van, vancomycin; Tet, tetracycline; Amp, ampicillin.
E. COLI AND ENTEROCOCCI IN GULL FAECES 875
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
Genomic characteristics of E. coli populations have also been
observed to vary temporally in human waste and faeces from
feral mice (Gordon 1997; Gordon et al. 2002). This variation
would be consistent with the concepts of E. coli resident and
immigrant strains suggested by earlier studies (Selander
et al. 1987).
Enterococci biochemical profiles support the concept of
bacterial population dynamics in gull faeces suggested by
the E. coli results. Most (23 of 25) June CHI Enterococcus
isolates had biochemical profiles identified as similar to
Ent. faecalis. In contrast, August CHI Enterococcus isolates
had different API� biochemical profiles than those of June
CHI isolates, and where species could be assigned, August
Enterococcus species were different from those obtained in
June. API biochemical tests alone cannot be used to assign
environmental enterococci isolates to species (Muller et al.
2001; Svec et al. 2002). The API tests have been developed
primarily for characterization of clinical isolates of entero-
cocci, and environmental isolates exhibit test responses
inconsistent with those established for clinical isolates of
the same species (Muller et al. 2001). Nevertheless, when
recently developed genotyping methods for enterococci have
been applied, biochemical test responses were consistent
within source-specific genotypes (ecovars; Svec et al. 2002)
and were useful in distinguishing closely related phylogenet-
ic groups of enterococci from less-related groups (Devriese
et al. 2002). In our study, antibiotic resistance patterns did
not closely parallel groups defined by biochemical test
response. Likewise, Muller et al. (2001) found little corre-
lation between antibiotic resistance patterns and species or
genotypes of enterococci isolated from forage grass. Never-
theless, some isolates in our study exhibited high-level
resistance to medically significant antibiotics.
The causes and ecological significance of population
variation in E. coli and enterococci in gull faeces remain to
be determined. Our results suggest the existence of ecovars
of both E. coli and enterococci in gull faeces, which might be
related to feeding ecology, age structure or colony charac-
teristics not determined in this study. In particular, gull diets
may be extremely variable. They are opportunistic feeders,
feeding on the nearest food supply (fish, worms, insects,
trash, etc.; Weseloh and Blokpoel 1979; Drury 1980; Hatch
1996) near the lakeshore, at landfills, in pastures or in city
parking lots. Early studies suggested that hosts are subject to
continuous immigration of E. coli strains from the environ-
ment, with food being a major source (Selander et al. 1987).
The abundance of Salmonella in gulls may be affected by
season and by age-specific differences in feeding ecology
(Hatch 1996). Studies performed on other animals have
shown that the intestinal microflora can be affected by small
changes in diet (Selander et al. 1987; Netherwood et al.1999; Souza et al. 1999; Leser et al. 2000). However, pigeons
have a very characteristic and host-specific enterococcal flora
(Baele et al. 2002). Further studies of E. coli and enterococci
population dynamics in gull faeces might lead to improved
understanding of gut ecology, and provide insight into the
significance of ecovars of these common bacterial genera.
The variation in faecal indicator bacteria populations seen
in this study is also significant in the context of current
efforts to determine the sources of faecal bacterial pollution
to ambient waters. First, our results suggest that the large
degree of variation in population characteristics of both E. coli
and enterococci in gull faeces will require extensive sampling
for adequate characterization. Second, our results may help
to better understand the variable success in correct classifi-
cation (ARCC) of bacteria with respect to source in other
studies (Hagedorn et al. 1999; Parveen et al. 1999; Dombek
et al. 2000; Carson et al. 2001). The variability in ARCC has
been similar regardless of method used. Therefore, it is likely
due to factors that are not method-related. Such factors may
include features of gut/faecal microbiology that exhibit
temporal, geographic or ecological variability. Intra-specific
variation in E. coli or Enterococcus genomic structure within a
given animal population may affect both the ARCC and the
reliability of various genomic typing procedures to correctly
classify these bacteria from various animal sources. Results of
this study suggest that variation in physiological and genomic
characteristics of E. coli and enterococci occurs at many
levels: within faecal samples, between faecal samples collec-
ted on the same date and between samples collected on
different dates. These variations are all important consider-
ations when building libraries to be used in faecal contam-
ination source determination.
ACKNOWLEDGEMENTS
We thank all those who helped in sample collection and
analyses including Joel Underwood from the US Geological
Survey, Lansing, MI; Maria Goodrich, from the US
Geological Survey, Porter, IN; and Brenda Berlowski and
Heather Gutzman from the US Geological Survey National
Wildlife Disease Center, Madison, WI. We would also like
to thank the City of Chicago for administrative, field and
technical support. This project was funded in part by the
City of Chicago, with contributions from the US Geological
Survey and the Michigan Great Lakes Protection Fund.
REFERENCES
Alderisio, K.A. and Deluca, N. (1999) Seasonal enumeration of fecal
coliform bacteria from the feces of ring-billed gulls (Larus
delawarensis) and Canada geese (Branta canadensis). Applied and
Environmental Microbiology 65, 5628–5630.
American Public Health Association (1998) Standard Methods for the
Examination of Water and Wastewater, 20th edn. Washington DC:
American Public Health Association.
876 L.R. FOGARTY ET AL.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
Baele, M., Devriese, L.A., Butaye, P. and Haesebrouck, F. (2002)
Composition of enterococcal and streptococcal flora from pigeon
intestines. Journal of Applied Microbiology 92, 348–351.
Carson, C.A., Shear, B.L., Ellersieck, M.R. and Asfaw, A. (2001)
Identification of fecal Escherichia coli from humans and animals
by ribotyping. Applied and Environmental Microbiology 67, 1503–
1507.
Devriese, L.A., Vancanneyt, M., Descheemaker, P., Baele, M.,
Landuyt, H.W.V., Gordts, B., Butaye, P., Swings, J. and
Haesebrouck, F. (2002) Differentiation and identification of Entero-
coccus durans, E. hirae, and E. villorum. Journal of Applied Micro-
biology 92, 821–827.
Dombek, P.E., Johnson, L.K., Zimmerley, S.T. and Sadowsky, M.J.
(2000) Use of repetitive DNA sequences and the PCR to differen-
tiate Escherichia coli isolates from human and animal sources. Applied
and Environmental Microbiology 66, 2572–2577.
Drury, W.H. (1980) Herring Gull, Hinterland Who’s Who, no. CW69-
4/10-2002E/. Ottawa, Canada: Canadian Wildlife Service, Envi-
ronment Canada.
Gordon, D.M. (1997) The genetic structure of Escherichia coli
populations in feral house mice. Microbiology 143, 2039–2046.
Gordon, D.M., Bauer, S. and Johnson, J.R. (2002) The genetic
structure of Escherichia coli populations in primary and secondary
habitats. Microbiology 148, 1513–1522.
Gould, D.J. and Fletcher, M.R. (1978) Gull droppings and their effects
on water quality. Water Research 12, 665–672.
Hagedorn, C., Robinson, S.L., Filtz, J.R., Grubbs, S.M., Angier, T.A.
and Reneau, R.B. Jr. (1999) Determining sources of fecal pollution
in a rural Virginia watershed with antibiotic resistance patterns in
fecal streptococci. Applied and Environmental Microbiology 65, 5522–
5531.
Harwood, V.J., Whitlock, J. and Withington, V. (2000) Classification of
antibiotic resistance patterns of fecal indicator bacteria by discrimi-
nant analysis: use in predicting the source of fecal contamination in
subtropical waters. Applied and Environmental Microbiology 66, 3698–
3704.
Hatch, J.J. (1996) Threat to public health from gulls. International
Journal of Environmental Health Research 6, 5–16.
Johnson, J.R. and O’Bryan, T.T. (2000) Improved repetitive-element
PCR fingerprinting for resolving pathogenic and nonpathogenic
phylogenetic groups within Escherichia coli. Clinical and Diagnostic
Laboratory Immunology 7, 265–273.
Jones, F., Smith, P. and Watson, D.C. (1978) Pollution of water supply
catchment by breeding gulls and the potential environmental health
implications. Journal of the Institution of Water Engineers and
Scientists 32, 469–482.
Jones, K. and Obiri-Danso, K. (1999) Non-compliance of beaches with
the EU directives of bathing water quality: evidence of non-point
sources of pollution in Morecambe Bay. Journal of Applied
Microbiology Symposium Supplement 85, 101S–107S.
Kaspar, C.W., Burgess, J.L., Knight, I.T. and Colwell, R.R. (1990)
Antibiotic resistance indexing of Escherichia coli to identify sources
of fecal contamination in water. Canadian Journal of Microbiology 36,
891–894.
Krumperman, P.H. (1983) Multiple antibiotic resistance indexing of
Escherichia coli to identify high-risk sources of fecal contamination of
foods. Applied and Environmental Microbiology 46, 165–170.
Lebuhn, M., Achouak, W., Scholter, M., Berge, O., Meier, H.,
Barakat, M., Hartmann, A. and Heulin, T. (2000) Taxonomic
characterization of Ochrobactrum sp. isolates from soil samples and
wheat roots, and description of Ochrobactrum tritici sp. nov. and
Ochrobactrum grignonense sp. nov. International Journal of Systematic
and Evolutionary Microbiology 50, 2207–2223.
Lemanceau, P., Corberand, T., Gardan, L., Latour, X., Laguerre,
Boeufgras, J. and Alabouvette, C. (1995) Effect of two plant species,
flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum
Mill.), on the diversity of soilborne populations of fluorescent pseu-
domonads. Applied and Environmental Microbiology 61, 1004–1012.
Leser, T.D., Lindecrona, R.H., Jensen, T.K., Jensen, B.B. and Møller,
K. (2000) Changes in bacterial community structure in the colon of
pigs fed different experimental diets and after infection with
Brachyspira hyodysenteriae. Applied and Environmental Microbiology
66, 3290–3296.
Levesque, B., Brousseau, P., Simard, P., Dewailly, E., Meisels, M. and
Joly, J. (1993) Impact of the ring-billed gull (Larus delawarensis) on
the microbiological quality of recreational water. Applied and
Environmental Microbiology 59, 1228–1230.
Levesque, B., Brousseau, P., Bernier, F., Dewailly, E. and Joly, J.
(2000) Study of the bacterial content of ring-billed gull droppings in
relation to recreational water quality. Water Research 34, 1089–1096.
Muller, T., Ulrich, A., Ott, E.-M. and Muller, M. (2001) Identification
of plant-associated enterococci. Journal of Applied Microbiology 91,
268–278.
National Committee for Clinical Laboratory Standards (2002) Per-
formance Standards for Antimicrobial Susceptibility Testing. 12th
Informational Supplement, National Committee for Clinical Labor-
atory Standards Document M100-S12, Wayne, Pennsylvania.
Netherwood, T., Gilbert, H.J., Parker, D.S. and O’Donnell, A.G.
(1999) Probiotics shown to change bacterial community structure in
the avian gastrointestinal tract. Applied and Environmental Microbio-
logy 65, 5134–5138.
Obiri-Danso, K. and Jones, K. (2000) Intertidal sediments as reservoirs
for hippurate negative camplybacters, salmonellae and faecal indi-
cators in three EU recognized bathing waters in North West
England. Water Research 34, 519–527.
Parveen, S., Murphree, R.L., Edmiston, L., Kaspar, C.W., Portier
K.M. and Tamplin, M.L. (1997) Association of multiple-antibiotic-
resistance profiles with point and nonpoint sources of Escherichia coli
in Apalachicola Bay. Applied and Environmental Microbiology 63,
2607–2612.
Parveen, S., Portier, K.M., Robinson, K., Edmiston, L. and Tamplin,
M.L. (1999) Discriminant analysis of ribotype profiles of Escherichia
coli for differentiating human and nonhuman sources of fecal
pollution. Applied and Environmental Microbiology 65, 3142–3147.
Parveen, S., Hodge, N.C., Stall, R.E., Farrah, S.R. and Tamplin, M.L.
(2001) Phenotypic and genomic characterization of human and
nonhuman Escherichia coli. Water Research 35, 379–386.
Rademaker, J.L.W. and de Bruijn, F.J. (1997) Characterization and
classification of microbes by rep-PCR genomic fingerprinting and
computer-assisted pattern analysis. In DNA Markers: Protocols,
Applications and Overviews ed. Caetano-Anolles, G. and Gresshoff,
P.M. pp. 151–171. New York: John Wiley.
Rademaker, J.L.W., Hoste, B., Louws Frank, J., Kersters, K., Swings,
J., Vauterin, L., Vauterin, P. and de Bruijn Frans, J. (2000)
E. COLI AND ENTEROCOCCI IN GULL FAECES 877
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878
Comparison of AFLP and rep-PCR genomic fingerprinting with
DNA–DNA homology studies: Xanthomonas as a model system.
International Journal of Systematic and Evolutionary Microbiology 50,
665–677.
Rose, J.B., Atlas, R.M., Gerba, C.P., Gilchrist, M.R., LeChevallier,
M.W., Sobsey, M.D. and Yates, M.V. (2001) Microbial Pollutants in
our Nation’s Water: Environmental and Public Health Issues. Wash-
ington DC: American Society for Microbiology.
Sauer, J.R., Hines, J.E. and Fallon, J. (2002) The North American
breeding bird survey, results and analysis 1966–2001. Version 2002.1.
USGS Patuxent Wildlife Research Center, Laurel, MD.
Selander, R.K., Caugant, D.A. and Whittam, T.S. (1987) Genetic
structure and variation in natural populations of Escherichia coli. In
Escherichia coli and Salmonella typhimurium, Cellular and Molecular
Biology, Vol. 2 ed. Neidhardt, F.C. pp. 1625–1645. Washington DC:
American Society for Microbiology.
Souza, V., Rocha, M., Valera, A. and Eguiarte, L.E. (1999) Genetic
structure of natural populations of Escherichia coli in wild hosts on
different continents. Applied and Environmental Microbiology 65,
3373–3385.
Svec, P., Devriese, L.A., Sedlacek, I., Baele, M., Vancanneyt, M.,
Haesebrouck, F., Swings, J. and Doskar, J. (2002) Characterization
of yellow-pigmented and motile enterococci isolated from intestines
of the garden snail Helix aspera. Journal of Applied Microbiology 92,
951–957.
USEPA (1986) Ambient water quality criteria for bacteria-1986.
EPA440/5-84-002. Washington DC: United States Environmental
Protection Agency.
USEPA (2000) Improved enumeration methods for the recreational
water quality indicators: enterococci and Escherichia coli. EPA/821/
R-97/004. Washington DC: United States Environmental Protec-
tion Agency.
Versalovic, J., Koeuth, T. and Lupski, J.R. (1991) Distribution of
repetitive DNA sequences in eubacteria and application to finger-
printing of bacterial genomes. Nucleic Acids Research 19, 6823–6831.
Wallace, J.S., Cheasty, T. and Jones, K. (1997) Isolation of Vero
cytotoxin-producing Escherichia coli O157 from wild birds. Journal of
Applied Microbiology 82, 399–404.
Weseloh, D.V. and Blokpoel, H. (1979). Ottawa, Canada: Canadian
Wildlife Service, Environment Canada.
Wiggins, B.A. (1996) Discriminant analysis of antibiotic resistance
patterns in fecal streptococci, a method to differentiate human and
animal sources of fecal pollution in natural waters. Applied and
Environmental Microbiology 62, 3997–4002.
Wiggins, B.A., Andrews, R.W., Conway, R.A., Corr, C.L., Dobratz,
E.J., Dougherty, D.P., Eppard, J.R., Knupp, S.R., Limjoco, M.C.,
Mettenburg, J.M., Rinehardt, J.M., Sonsino, J., Torrijos, R.L. and
Zimmerman, M.E. (1999) Use of antibiotic resistance analysis to
identify nonpoint sources of fecal pollution. Applied and Environ-
mental Microbiology 65, 3483–3486.
878 L.R. FOGARTY ET AL.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 865–878