Antibiotic resistant enterococci and staphylococci isolated from flies collected near confined poultry feeding operations potx

Antibiotic resistant enterococci and staphylococci isolated from
flies collected near confined poultry feeding operations
Jay P. Graham
⁎
, Lance B. Price, Sean L. Evans, Thaddeus K. Graczyk, Ellen K. Silbergeld
Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Sciences, Division of Environmental Health
Engineering, Baltimore, MD 21205, USA
ARTICLE DATA ABSTRACT
Article history:
Received 29 July 2008
Received in revised form
17 November 2008
Accepted 25 November 2008
Use of antibiotics as feed additives in poultry production has been linked to the presence of
antibiotic resistant bacteria in farm workers, consumer poultry products and the environs of
confined poultry operations. There are concerns that these resistant bacteria may be
transferred to communities near these operations; however, environmental pathways of
exposure are not well documented. We assessed the prevalence of antibiotic resistant
enterococci and staphylococci in stored poultry litter and flies collected near broiler chicken
houses. Drug resistant enterococci and staphylococci were isolated from flies caught near
confined poultry feeding operations in the summer of 2006. Susceptibility testing was
conducted on isolates using antibiotics selected on the basis of their importance to human
medicine and use in poultry production. Resistant isolates were then screened for genetic
determinants of antibiotic resistance. A total of 142 enterococcal isolates and 144
staphylococcal isolates from both fly and poultry litter samples were identified.
Resistance genes erm(B), erm(A), msr(C), msr(A/B) and mobile genetic elements associated
with the conjugative transposon Tn916, were found in isolates recovered from both poultry
litter and flies. Erm(B) was the most common resistance gene in enterococci, while erm(A)
was the most common in staphylococci. We report that flies collected near broiler poultry
operations may be involved in the spread of drug resistant bacteria from these operations
and may increase the potential for human exposure to drug resistant bacteria.

© 2008 Elsevier B.V. All rights reserved.
Keywords:
Antibiotic resistance
Enterococci
Flies
Poultry litter
Staphylococci
1. Introduction
There is growing public health concern over the contribution of
agricultural antibiotic use to the global rise of drug resistant
bacteria (Erb e t al., 2007 ; Levy and Marshall, 2004). The U.S. raises
approximately 8.7 billion broiler chickens annually, resulting in
an estimated 13–26 million metric tons of poultry litter (i.e.,
excreta, feathers, spilled feed, bedding material, soil and dead
birds) (Moore et al., 1995; Paudel et al., 2004). Antibiotics are
permittedas additivestofeed or waterinthe U.S.(NRC, 1999)and
it is estimated that nearly 80% of poultry units in the U.S. use
antibiotics in feed (Silbergeld et al., 2008). Poultry litter has been
found to contain large amounts of antibiotic resistant bacteria
and resistance genes associated with the use of antibiotics in
poultry production (Nandi et a l., 2004). This has raised concern
for environmental dispersal of antibiotic resistance. In this
study, we report for the first time that houseflies may also
participate inthe dispersion of antibiotic resistance from poultry
houses into the environment. Houseflies have practically
unconstrained access to this litter, both through entrance into
SCIENCE OF THE TOTAL ENVIRONMENT XX (2009) XXX– XXX
Abbreviations: ATCCAmerican Type Culture Collection; CLSIClinical and Laboratory Standards Institute; E.Enterococcus; MICminimum
inhibitory concentration; PCRpolymerase chain reaction; ORFopen reading frame; rRNAribosomal ribonucleic acid; S.Staphylococcus.
⁎ Corresponding author. Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Science s, Division of

Environmental Health Engineering, 615 N. Wolfe St., Room E6642, Baltimore, MD 21205, USA. Tel.: +1 443 286 8335; fax: +1 410 955 9334.
E-mail address: (J.P. Graham).
0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.11.056
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confined poultry feeding operations, Sci Total Environ (2009), doi:10.1016/j.scitotenv.2008.11.056
poultry houses as well as access to poultry waste stored onsite in
open sheds. Prior to land application, poultry litter is generally
piled between 1 and 4 m deep andstored in open sheds until it is
applied to land as a soil amendment. Populations of houseflies
are known to be significantly increased within distances of upto
7 km away from poultry operations (Winpisinger et al., 2005).
Synanthropic flies have evolved to live in proximity to
humans and have been found to carry a number of different
pathogenic microorganisms, including viruses and bacteria, and
can play an important role in the epidemiology of infections in
humans (Likirdopulos et al., 2005; Macovei and Zurek, 2006;
Nichols, 2005). Flies have been implicated in the spread of a
number of bacterial infections, such as: enteric fever, cholera,
shigellosis, salmonellosis, and campylobacteriosis (Fotedar
et al., 1992; Nichols, 2005). There is recent concern that flies
may also contribute to the spread of avian influenza. A study in
Denmark found that as many as 30,000 flies may enter a broiler
facility during a single flock rotation in the summer months
(Hald et al., 2004). In Japan, researchers reported that flies
captured in proximity to broiler facilities during an outbreak of
highly pathogenic avian influenza in Kyoto, Japan in 2004, were

found to carry the same strains of H5N1 influenzavirus as found
in the chickens of the infected poultry farm (Sawabe et al., 2006).
The pathway of transfer is likely to occur as flies feed on excreta
and decomposing carcasses, which results in ingestion of the
bacteria or surface contamination of their feet, legs, proboscis,
and wings. The flies can then mechanically transmit micro-
organisms through physical contact or may defecate or
regurgitate bacteria from the gut onto food or other fomites
(Nichols, 2005). The quantity and type of microorganisms flies
carry are inextricably linked to the presence of these same
organisms in the excreta and other wastes upon which flies
develop and feed (Nichols, 2005).
The design and operational requirements of large scale
broiler poultry production result in many obstacles to biocon-
tainment (i.e., efforts to limit the dissemination of microbes
from operations) (Graham et al., 2008). Ventilation rates from
these houses are very high, owing to the need to prevent
overheating for the 20–75,000 birds confined to a single house.
Further, owing to methods of waste storage at farms, there is a
large amount of fresh and stored poultry litter available outside
the houses, which can serve also as a substrate for development
of fly populations and a readily available source of food.
Because antibiotic resistant enterococci and staphylococci
have been isolated from poultry litter (Hayes et al., 2004; Lu
et al., 2003; Simjee et al., 2007), we tested the hypothesis that
flies may transfer these resistant pathogens, as well as
resistance determinants, into the environment of local com-
munities. This mode of inter-ecosystem spread has not been
previously investigated.
The current study is the first to assess resistance pheno-

types and resistance genes in Enterococcus spp. and Staphylo-
coccus spp. in both litter and flies collected near U.S. confined
poultry feeding operations.
2. Methods
Sampling was carried out on the Delmarva Peninsula of the
United States (region comprising parts of Delaware, Maryland,
and Virginia), one of the most heavily concentrated areas of U.S.
poultry production (Fig. 1), producing nearly 600 million chick-
ens each year (nearly 7% of U.S. production). It is also an area
experiencing rapid development and increased human popula-
tion density.Sussex County, Delaware, wherenearly 300 million
chickens were produced last year, experienced a 15% increase in
its human population between 2000 and 2006 (Delaware
Population Consortium, 2002).
2.1. Poultry litter collection
Poultry litter samples were collected from three conventional
poultry farms that raised the birds under contract for two
major producers. Litter samples were collected from three
conventional broiler chicken farms over a period of 120 days
(collected at Days: 0, 10, 20, 30, 60, 90, 120) in the summer of
2006. The first sampling visit at each farm occurred after the
chickens were removed for processing, at which time the
houses were decrusted, that is, removing the top 25–50 cm of
poultry litter from the poultry house floor. This waste material
was stored on-site in one large pile between 1 to 3 m high in a
two-walled shed with a roof. No additional litter was added
during the study period, nor were any chemicals added. A
composite sample of four grab samples (~1 kg) from each litter
pile was aseptically collected at each visit and placed in sealed
plastic bags for transport in a cooler with ice to the laboratory.

Samples were analyzed within 24 h of collection. All three
farmers reported that no recognized disease outbreaks had
occurred during the flock cycle such that no therapeutic drug
use was applied, but no specific information on antibiotic feed
additives was available as this is considered confidential
business information by the producers (Graham et al., 2007).
Each poultry litter sample was mixed in the sealed plastic
bag by vigorously agitating the bag by hand for 1 min. Five
grams of litter were then placed in 45 ml of 0.1% peptone water
in a sterile 50 ml polypropylene conical tube, and vortexed for
1 min (Islam et al., 2004). The sample was allowed to settle for
15 min. Three serial dilutions (1:10) were prepared from each
sample using 0.1% peptone water, and 0.1 ml portions of each
dilution were plated in triplicate onto standard BBL Enter-
ococcosel agar (Becton Dickinson, Cockeysville, MD, USA) and
Staphylococcus agar (US Biological, Swampscott, MA, USA).
Samples were plated on agar supplemented with antibiotics at
break point concentrations described below. Samples were
incubated for 24 h at 37 °C, and unique black enterococcal and
yellow/white staphylococcal colonies were selected. Isolates
were purified twice on the same medium on which they were
isolated. All isolates were stored in a 20% glycerol tryptic soy
broth at − 80 °C until testing for antibiotic susceptibility.
2.2. Fly collection and bacterial isolation
Flies were caught using Victor Fly Magnet
®
Traps at the same
time period as when the last poultry litter samples were
collected (i.e., day 120). A total of eight fly traps were set in
accessible locations within 15–100 m of poultry farms, and

placed approximately 2 m off the ground. Although the fly traps
were not set near the farms where litter samples were collected,
we hypothesized that similar resistance patterns among the fly
and litter isolates would be observed. The traps were collected
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Fig. 1 – Map of study area (Delmarva Peninsula) with sample locations and resistance genes or mobile genetic elements recovered from bacterial isolates. The exact location of
farms was not provided in order to maintain farmer anonymity.
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36 h after set up, and transported to the laboratory and stored at
4 °C. Flies caught in each trap were treated as one composite
sample because of likely contact and mixing, and were analyzed
within 24 h of collection. An external wash of the flies was
carried out as follows: flies were placed into a plastic tube with
50 ml of eluting buffer, consisting of 0.1% Tween80, 0.1% sodium
dodecyl sulfate, 0.001% anti-foam, and phosphate-buffered
saline, and then gently vortexed for 1 min (Graczyk et al.,
1999). One ml of the eluant was then aseptically transferred in a
15 ml plastic tube with 10 ml of tryptic soy broth for a 24 h
enrichment. Following this exterior wash, a homogenized
sample of the flies (i.e., internalized bacteria) was made as
follows: flies from each trap were placed together in an
Eppendorf tube (BWR, Piscataway, NJ) with 50 ml of phos-
phate-buffered saline and were macerated with a glass
rod for 1 min. One ml of the homogenate was then enriched

as described above. Following the enrichment, 0.1 ml portions of
the enriched samples were plated onto standard BBL Enter-
ococcosel agar (Becton Dickinson, Cockeysville, MD, USA) and
Staphylococcus agar (US Biological, Swampscott, MA, USA).
2.3. Isolation of antibiotic resistant bacteria
Samples of the enrichment media were plated on agar supple-
mented with selected antibiotics in order to increase the like-
lihood of detecting resistant enterococci and staphylococci
Table 1 – List of positive controls and DNA oligonucleotides used as primers in PCR reactions
Genus/species (single/
multiplex PCR)
Positive
control
Direction Sequence (5′–3′) Annealing
temp (°C)
Product
size (bp)
Reference
Enterococci
a
F TCAACCGGGGAGGGT 60 733 Deasy et al.
(2000)R ATTACTAGCGATTCCGG
E. faecalis
a
ATCC
29212
F TCAAGTACAGTTAGTCTTTATTAG 54 941 Dutka-Malen
et al. (1995)R ACGATTCAAAGCTAACTGAATCAGT
E. faecium
a

ATCC
19434
F TTGAGGCAGACCAGATTGACG 54 658 Dutka-Malen
et al. (1995)R TATGACAGCGACTCCGATTCC
E. casseliflavus
a
ATCC
49605
F CGGGGAAGATGGCAGTAT 54 484 Kariyama
et al. (2000)R CGCAGGGACGGTGATTTT
E. gallinarum
a
ATCC
700425
F GGTATCAAGGAAACCTC 54 822 Kariyama
et al. (2000)R CTTCCGCCATCATAGCT
Staphylococci F GGCCGTGTTGAACGTGGTCAAATCA 55 370 Morot-Bizot
et al. (2004)R TIACCATTTCAGTACCTTCTGGTAA
S. aureus ATCC
43300
F AATCTTTGTCGGTACACGATATTCTTCACG 55 108 Morot-Bizot
et al. (2004)R CGTAATGAGATTTCAGTAGATAATACAACA
S. xylosus ATCC
29971
F AACGCGCAACGTGATAAAATTAATG 55 539 Morot-Bizot
et al. (2004)R AACGCGCAACAGCAATTACG
S. epidermidis ATCC
49461
F ATCAAAAAGTTGGCGAACCTTTTCA 55 124 Morot-Bizot
et al. (2004)R CAAAAGAGCGTGGAGAAAAGTATCA

S. saprophyticus ATCC
49453
F TCAAAAAGTTTTCTAAAAAATTTAC 55 221 Morot-Bizot
et al. (2004)R ACGGGCGTCCACAAAATCAATAGGA
a
Multiplex PCR was used for all of the Enterococci primers.
Table 2 – List of PCR primers used in the amplification of resistance genes in isolates of enterococci and staphylococci
Resistance gene/
determinant
GenBank
access. no
Direction Primer sequence (5′–3′) Annealing
temp (°C)
Product
size (bp)
Reference
erm(A) K02987 F TCAAAGCCTGTCGGAATTGG 52 441 Jensen et al.
(2002)R AAGCGGTAAACCCCTCTGAG
erm(B) AF406971 F GAAAAGGTACTCAACCAAATA 52 639 Sutcliffe et al.
(1996)R AGTAACGGTACTTAAATTGTTTAC
erm(C) J01755 F ATCTTTGAAATCGGCTCAGG 52 294 Sutcliffe et al.
(1996)R CAAACCCGTATTCCACGATT
vat(D) L12033 F GCTCAATAGGACCAGGTGTA 52 271 Soltani et al.
(2000)R TCCAGCTAACATGTATGGCG
vat(E) AF139725 F ACTATACCTGACGCAAATGC 52 511 Soltani et al.
(2000)R GGTTCAAATCTTGGTCCG
msr(C) AF13494 F TAT AAC AAA CCT GCA AGT TC 55 1,040 McDermott et al.
(2005)R CTT CAA TTA GTC GAT CCA TA
msr(A/B) AJ243209 F GCAAATGGTGTAGGTAAGACAACT 55 350 Wondrack et al.
(1996)R ATCATGTGATGTAAACAAAAT

int (Tn916/Tn1545) NC006372 F GCGTGATTGTATCTCACT 50 1,046 Macovei and
Zurek (2006)R GACGCTCCTGTTGCTTCT
ORF13 (Tn916) NC006372 F GGCTGTCGCTGTAGGATAGAG 50 589 Macovei and
Zurek (2006)R GGGTACTTTTAGGGCTTAGT
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strains among an expected mix of resistant and susceptible
strains within the litter sample. All but one of the following
antibiotics (i.e. vancomycin) or similar analogs were selected
based on their reported use in poultry production and added to
agar (concentrations added to enterococcosel a nd staphylococcus
agar are indicated respectively): ciprofloxacin (2 μg/ml, 2 μg/ml),
clindamycin (1 μg/ml, 2 μg/ml), tetracycline (8 μg/ml, 8 μg/ml),
vancomycin (16 μg/ml, 1 6 μg/ml), erythromycin (4 μg/ml, 4 μg/ml),
quinupristin-dalfopristin (2 μg/ml, 2 μg/ml), penicillin (8 μg/ml,
0.125 μg/ml), and g entamicin ( 500 μg/ml in enterococcosel only).
Samples were incubated for 24 h a t 37 °C, and representative
unique colonies based on colony morpholog y were se lected.
Isolates were purified and stored as described previously. The
antibiotic quinupristin-dalfopristin is an analog of v irginiamycin,
an antibiotic used in poult ry production. Both qui nupristin-
dalfopristin and v irginiamycin are i n the sam e class o f antibiotics.
Table 3 – Characteristics of samples of flies and stored poultry litter
Fly
samples
Number
of flies
Distance in meters/

direction from nearest
poultry farm
Number of
enterococcal isolates
characterized
Number of
staphylococcal
isolates characterized
MDR enterococci
=2 drugs
≥ 3 drugs
MDR staphylococci
=2 drugs
≥ 3 drugs
Trap 1 3 60 m 1 4 + −
Southeast −−
Trap 2 28 100 m 8 4 + +
East + −
Trap 3 6 30 m 3 4 + +
South ++
Trap 4 8 20 m 2 3 + −
Southeast −−
Trap 5 7 15 m 3 4 + −
Southeast −−
Trap 6 28 50 m 7 1 + −
Southeast + −
Trap 7 140 100 m 12 5 + +
South + −
Trap 8 42 30 m 0 4 −−
Southeast −−

Poultry litter
samples
Number
of samples
Number of
enterococcal isolates
characterized
Number of
staphylococcal
isolates characterized
MDR enterococci
=2 drugs
≥ 3 drugs
MDR staphylococci
=2 drugs
≥ 3 drugs
Farm A 7 36 35 + +
++
Farm B 5 25 30 + +
++
Farm C 7 45 50 + +
++
Fig. 2 – Percent of recovered enterococcal isolates phenotypically resistant to antibiotics. Multi-drug resistance (MDR) indicates
resistance to two or more drugs. (cip – ciprofloxacin; clin – clindamycin; ery – erythromycin; pen – penicillin; q-d – quinupristin-
dalfopristin; tet – tetracycline; van – vancomycin; MDR – multi-drug resistant).
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2.4. Species identification

PCR was used to confirm the identities of the isolates to the
genus level (Table 1). Single PCR and Multiplex PCR were used
to identify four common species of enterococci (E. faecium,
E. faecalis, E. gallinarum, and E. casseliflavus) and four common
species of staphylococci (S. aureus, S. xylosus, S. saprophyticus,
and S. epidermidis). ATCC strains used as positive controls and
primer sequences are provided in Table 1.
2.5. Antibiotic resistance screening
Phenotypic antibiotic resistance was defined by minimal
inhibitory concentrations (MICs) which were determined using
the agar dilution method on Mueller–Hinton agar (Becton
Dickinson, Massachusetts) using Enterococcus faecalis ATCC
29212, Enterococcus faeciumATCC 19434, and Staphylococcus aureus
ATCC 43300 strains according to CLSI guidelines (CLSI, 2005).
The dilution ranges in μg/ml and resistance breakpoints were as
follows (note: breakpoints for enterococci and staphylococci are
the same unless otherwise stated): ciprofloxacin (0.12–8, 4),
clindamycin (0.5–8, 2 for enterococci and 4 for staphylococci),
tetracycline (1–32, 16), v a ncomycin (0.5–64, 3 2 for ente rococci and
16 for staphylococci), erythromycin (0.13–16, 8), quinupristin-
dalfopristin (0.025–8, 4), penicillin (0.5–32, 16 for enterococci and
0.25 for staphylococci), and gentamicin (500–1000, 500 for
enterococci). For staphylococci, no CLSI breakpoints have been
establish ed for a number of drugs ( e.g. clindamycin , penicillin or
vancomycin) and breakpoints as described by Aarestrup et al.
(2000) wereused. When s trains of identical species from t he same
farm having similar antibiograms (i.e. within two d ilutions) were
found, only one isolate was used for the analysis – this was done
to ensure that t he same i solate was not counted more than once.
2.6. Screening for resistance genes

For each isolate exhibiting phenotypic resistance to eryth-
romycin, quinupristin-dalfopristin, or tetracycline, the
bacteria were harvested and cell walls were digested with
lysozyme and proteins were subsequently digested with
proteinase k and sodium dodecyl sulfate. DNA was isolated
using a phenol-chloroform extraction and isopropyl alcohol
precipitation method (Sutcliffe et al., 1996) and was quantified
using a NanoDrop
®
ND-1000 UV–V is Spectrophotometer
(Wilmington, DE, USA). Each DNA sample was standardized
to a final concentration of 20 ng/μl. Single PCR was used
to screen isolat es that were phenotypically resistant to
macrolides, lincosamides, tetracyclines, or streptogramins.
Detection of the rRNA methylase genes (erm(A), erm(B), erm(C)),
the acetyl transferase genes (vat(D) and vat(E)) , and the ABC
porter genes (msr(A/B)and ms r(C)) was carried out using primers
and PCR conditions previously described (Table 2). The PCR
assay mix (total volume of 12.5 μl) included 1 U Takara Taq
HotStart DNA Polymerase and 10X PCR Buffer (Takara Bio Inc,
Otsu,Shiga,Japan),0.5μM of ea ch p rimer, 200 μMofeachdNTP
and 40 ng of genomic DNA (i.e. 2 μl of sample). Most resistance
genes were amplified with an initial denaturing cycle at 95 °C
for 5 min followed by 25 cycles of 94 °C for 45 s, 52 °C for 45 s,
and 72 °C for 1 min, with a final extension step at 72 °C for
10 min. Genes, msr(C) and msr(A/B) were amplified under
different conditions: an initial denaturing cycle at 95 °C for
5 min was followed by 25 cycles of 93 °C for 30 s, 55 °C for
2 min, and 72 °C for 1.5 min, with a final extension step at
72 °C for 10 min. PCR products were run on a 2% agarose

gel. The class 1 integrase gene was used for detection of the
Tn916/Tn 1545 conjugative transposon family and the open
reading frame gene (ORF1 3) was used for specific detection of
Tn916 (Macovei and Zurek, 2006).
3. Results
Trapped flies were identified as members of Muscidae (house-
flies) and Calliphoridae (blow flies and bottle flies) families.
The number of flies and number of bacterial isolates recovered
varied across the traps shown in Table 3.
Fig. 3 – Percent of recovered staphylococcal isolates phenotypically resistant to antibiotics. Multi-drug resistance (MDR)
indicates resistance to two more drugs. Only antibiotics with CLSI established breakpoints are presented. (cip – ciprofloxacin;
ery – erythromycin; q-d – quinupristin-dalfopristin; tet – tetracycline; MDR – multi-drug resistant).
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Resistant enterococci and staphylococci persisted in the
litter piles throughout the 120 da y study period. However,
resistant enterocococci were isolated at fewer farms at later
sampling events. For example, resistance to four drug s was
not observed in enterococc i after day 60. T his was not the
case, however, for staphylococci, where drug resistance to
more than three drugs was observed from samples collected
at day 120. After removing duplicate isolates (described in
Met hods), a total of 106 enterococcal and 115 staphylococcal
isolates were characterized from poultry litter samples,
while 36 enterococcal and 29 staphylococcal isolates were
characterized from fly samples. In both the fly and poult ry
litter samples, Enterococcus faecalis represented the majority
of the enterococcal species (70% in litter and 87% in flies).

Most staphylococcal isolates did not correspond to the
species primers in our study (Table 1) and were characterized
to th e genus level only, with the exception of s even isolates
of S. xylosus, five isolates of S. epidermidis, and three isolates
of S. aureus. Approximately two-thirds of staphylococci and
enterococci isolated from flies were o btained from the ho-
mogenized samples (i.e., internalized bacter ia), and approxi-
mately one-third were obtained from exterior washes.
The results of resistance testing are shown in Figs. 2 and 3
(note: isolates were recovered from both antibiotic-amended
and non-amended plates). Resistance to clindamycin was the
most common resistance phenotype in enterococcal isolates
Table 4 – Characteristics of individual isolates positive for resistance genes and/or mobile genetic elements
Genus/species Sample location Phenotypic resistance
a
Mobile element Resistance genes
Enterococcus faecium
Farm C clin
r
, ery
r
, q-d
r
, tet
r
Tn916 erm(B)
Farm C clin
r
, ery
r

erm(B)
Farm C clin
r
, ery
r
, q-d
r
erm(B), vat(E), msr(C)
Farm C clin
r
, ery
r
, pen
r
, q-d
r
erm(A)
Farm C clin
r
, ery
r
, q-d
r
, tet
r
Tn916
Farm C clin
r
, tet
r

msr(C)
Trap 2 clin
r
, ery
r
, q-d
r
Tn916
Trap 3 clin
r
, ery
r
, q-d
r
, tet
r
erm(B)
Trap 6 clin
r
, tet
r
Tn916 msr(C)
Trap 7 clin
r
, q-d
r
, tet
r
Tn916 msr(C)
Enterococcus faecalis

Farm A clin
r
, ery
r
, q-d
r
, tet
r
Tn916 erm(B)
Farm B clin
r
, ery
r
, q-d
r
erm(B)
Farm B clin
r
, ery
r
, q-d
r
, tet
r
Tn916 erm(B)
Farm A clin
r
, tet
r
Tn916

Farm A clin
r
, ery
r
, q-d
r
, tet
r
Tn916 erm(B)
Farm B clin
r
, ery
r
, q-d
r
erm(B)
Farm C clin
r
, ery
r
, tet
r
Tn916 erm(B)
Farm A clin
r
, ery
r
, q-d
r
, tet

r
Tn916 erm(B)
Farm C clin
r
, q-d
r
Tn916 erm(B)
Trap 1 clin
r
, q-d
r
erm(B)
Trap 2 clin
r
, q-d
r
erm(B)
Trap 2 clin
r
, ery
r
, q-d
r
, tet
r
erm(B)
Trap 2 clin
r
, ery
r

, q-d
r
, tet
r
Tn916 erm(B)
Trap 3 clin
r
, ery
r
, q-d
r
, tet
r
erm(B)
Trap 6 clin
r
, pen
r
, tet
r
Tn916 erm(B)
Trap 6 clin
r
, ery
r
, q-d
r
, tet
r
erm(B)

Trap 7 clin
r
, ery
r
, q-d
r
erm(B)
Trap 7 clin
r
, q-d
r
, tet
r
Tn916
Trap 7 clin
r
, ery
r
, q-d
r
, tet
r
Tn916 erm(B)
Trap 7 clin
r
, ery
r
, q-d
r
, tet

r
Tn916 erm(B)
Staphylococcus spp.
Farm A ery
r
, tet
r
erm(A)
Farm B ery
r
msr(A/B)
Farm B clin
r
, ery
r
erm(A), erm(C)
Farm B ery
r
erm(A)
Farm B ery
r
, tet
r
erm(A)
Farm C ery
r
msr(A/B)
Farm C ery
r
erm(A), msr(A/B)

Farm A ery
r
msr(A/B)
Farm C q-d
r
erm(A)
Trap 5 ery
r
msr(A/B)
Note: only isolates exhibiting phenotypic resistance to erythromycin, quinupristin-dalfopristin, or tetracycline were screened for resistance genes.
a
Phenotypic resistance: e ry
r
– erythromycin resistant; q-d
r
– quinupristin-dalfopristin resistant; tet
r
– tetracycline resistant; clin
r
– clindamycin resistant;
pen
r
– penicillin res istant.
7SCIENCE OF THE TOTAL ENVIRONMENT XX (2009) XXX– XXX
ARTICLE IN PRESS
Please cite this article as: Graham JP et al., Antibiotic resistant enterococci and staphylococci isolated from flies collected near
confined poultry feeding operations, Sci Total Environ (2009), doi:10.1016/j.scitotenv.2008.11.056
from both fly and poultry litter samples. Resistance to the
lincosamide class of antibiotics (which includes clindamycin)
has been reported to be an intrinsic trait that is relatively

common in E. faecalis (Hayes et al., 2004). Among the
enterococcal isolates recovered from flies, resistance was
more common for quinupristin-dalfopristin (94%), erythromy-
cin (42%) and tetracycline (39%) than in isolates of poultry
litter origin (Fig. 2). Very little resistance to penicillin and
ciprofloxacin was observed for enterococcal isolates from
either flies or litter ( Fig. 2). Further, no enterococcal isolates
were found to be resistant to vancomycin.
In staphylococcal isolates, phenotypic resistance to ery-
thromycin was relatively more common in litter isolates (57%)
than in isolates from flies (19%). The percentage of staphylo-
coccal isolates resistant to quinupristin-dalfopristin and
tetracycline was also higher in litter (30%) as compared to
flies (10%). There are no established breakpoints for clinda-
mycin and penicillin; however, approximately 90% of isolates
from either flies or litter had an MIC value of less than 0.25 μg/
ml. One staphylococcal isolate from poultry litter exhibited
high level resistance to vancomycin (64 μg/ml).
Erm(B) was the resistance gene most commonly found in
enterococci in both flies and poultry litter isolates (Table 4).
Isolates found to carry erm(B) were also likely to be resistant
to quinupristin-dalfopristin, erythromycin and clindamycin.
This gene alters a site in 23S rRNA common to the binding of
macrolides, lincosamides and streptogramin
B
antibiotics
(Sutcliffe et al., 1996). The enterococcal gene, msr(C) was
observed in two isolates from poultry litter and two i solates
from fly samples. The nearly homologous staphylococcal
gene, msr(A /B), was observed in four isolates from poultry

litter and one isolate from fly samples. The msr genes encode
an ABC porter for macrolide and streptogramin
B
antibiotics.
The ORF13 gene, which is associated with the conjugative
transposon Tn916, was found in nine enterococcal isolates
from poultry litter and eight from fly isolates; Tn916 repre-
sents a family of transposons commonly found to transfer
antibiotic resistance genes. The combination of ORF13 gene
and int gene, associated with Tn1545/916, were recovered
from four enterococcal isolates from poultry litter and six from
fly isolates, all of which also contained the erm(B) gene
(Table 4). Two fly isolates from traps 6 and 7 placed in
proximity, also contained the msr(C) gene in combination with
Tn916.
The percentage of phenotypically resistant enterococcal
isolates – resistant to erythromycin, quinupristin-dalfopristin,
or tetracycline – positive for resistance determinants was
nearly identical among fly and poultry litter isolates (Fig. 4).
4. Discussion
This study strongly suggests that flies in intensive poultry
production areas, such as the Delmarva Peninsula, can
disperse antibiotic resistant bacteria in their digestive tracts
and on their exterior surfaces. Dispersion of resistant bacteria
from poultry farms by flies could contribute to human
exposures, although at present it is difficult to quantify
the contribution of flies. Flies may also transfer bacteria
from fields amended with poultry waste. Fly populations have
been found to be higher near poultry farms as compared to
nearby rural settings (Winpisinger et al., 2005). Although

individual flies can travel as far as 20 miles, the majority of
the species found in traps in this study generally do not travel
more than 2 miles and their movement is oriented toward
readily available food sources (Graczyk et al., 1999; Sawabe et
al., 2006).
Six of the eight classes of antibiotics screened in this study
[penicillin, tetracyclines, macrolides, lincosamides, aminogly-
cosides, and streptogramins] are used in poultry production,
while fluoroquinolones were used until 2005 (Florini et al.,
2005; Price et al., 2007). All of these drugs are categorized by the
U.S. Food and Drug Administration as critically or highly
important to human medicine (USFDA, 2003). Staphylococcal
Fig. 4 – Percentage of enterococci isolates (phenotypically resistant to either erythromycin, quinupristin-dalfopristin, or
tetracycline) positive for resistance determinants.
8 SCIENCE OF THE TOTAL ENVIRONMENT XX (2009) XXX– XXX
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Please cite this article as: Graham JP et al., Antibiotic resistant enterococci and staphylococci isolated from flies collected near
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infections are often treated with penicillins, macrolides,
lincosamide, aminoglycosides, and streptogramins, while
enterococcal infections are usually treated with penicillins,
aminoglycosides, tetracyclines and streptogramins (Bartlett
et al., 2005). Of concern, streptogramins, which have been used
in animal husbandry for near ly 30 years, were recently
approved for treating patients with vancomycin resistant E.
faecium or methicillin-resistant Staphylococcus aureus ( Jensen
et al., 2002; McDermott et al., 2005).
Enterococci resistance to streptogramins (quinupristin-
dalfopristin), were found in both litter and flies. Quinupristin-
dalfopristin resistant enterococci in our study commonly had erm (A)

and erm(B) resistance genes. Streptogramin
A
(i.e. dalfopristin)
resistance in E. faecium, isolated from the poultry environment,
has been found to be highly associated with the vat(E) gene,
while Streptogramin
B
(i.e. quinupristin) resistance has been
linked to the erm(B) gene (Jensen et al., 2002). The emergence of
streptogramin-resistant E. faecium,associatedwiththeerm
genes conferring resistance to streptogramin
B
,andvat genes
conferring high-level resistance to streptogramin
A
, is a serious
public health concern, and is thought to be a consequence of
the use of virginiamycin for growth promotion over the past
30 years (Smith et al., 2003). The absence of vancomycin
resistant enterococci in our study was not a surprise, given
that vancomycin has never been approved for use in U.S
food animal production. In contrast, vancomycin resistant
enterococci have been frequently reported in European studies,
where avoparcin (an analog of vancomycin) was used in animal
feeds until 1997 (Aarestrup et al., 2001). It was surprising,
however that we cultured one staphylococcal isolate from the
poultry litter that exhibited high-level resistance to vancomycin
(N 64 μg/ml).
Most conjugative transposons of the Tn916 family encode
resistance to tetracycline or minocycline a lone, and tetracy cline

resistance is now relatively common. Although increased
prevalence o f resistance and the availability of a variety of other
broadly active antibiotics have reduced the importance of
tetracycline as a therapeutic alternative, it remains a first- and
second-line t reatment for many urogenit al infecti ons (Rice, 1 998).
The clustering of resistance genes on the same t ransposable
elements can affect the persistence o f antibiotic re sistance, such
that elimina ting only one an tibioti c may not reduce t he pre-
valence o f the cluster. The er m(B) gene, for example, i s c ommonly
linked with Tn154 5/Tn 916, w hich encodes tetracycline resistance
and predominates i n clinically i mportant Gram-positive ba cteria
(Clewe ll et al., 1995; Rice, 1998). The continued dissemination of
mobile genetic elements that have broad host-range, such as
Tn916 family, which includes Tn 1545, in the microbial environ-
ment is a serious problem.
One o f t he li mitations of this study is that a small number of
sampling sites were used and fly and litter samples were not
collected from the same sites. This may account for the
differences observed between the p henotypic resi stance patte rns
of is olates from flies and litter. However, be cause flies can travel
as much as 20 miles, it is not possible to ascertain associ-
ations between a specific sample of flies and a specific farm.
An additional l imitation was the limited c oagulase-negative
Staphylococcus species characterized in the analyses. Other
species, such as S. sciuri, S. lentus,andS. simulans would have
been likely candidates, as s hown by Sim jee et al. (2007) in a study
of poultry litter in Georgia. Additionally , no control sites were
used. A proper control s ite would have been difficult to define in
this setting as poultry production occurs throughout the
Delmarva Peninsula, as well as land amendment with poultry

wastes, and flies can potentially travel long distances. Another
limitation w as that we could not obta in data on antibiotic use at
any of the farms sampled since this information is not publicly
available in the U.S. (Mellon et al., 2001). There is a lack of
definitive i nformation on the overallv olume of antibiotics used as
feed additives, and there are obstacles to this information since
feed formulations are considered confidential b usiness informa-
tion under U.S. law. Nonetheless, our data are consistent with
studies highlighting the prevalence of resistant enterococci and
staphylococci in the poultry environment (Hayes et al., 2004;
Lu et al., 2003).
5. Conclusions
The results of this study illustrate the persistence of resistant
bacteria in the environment, and highlight the reservoir of
resistance associated with the use of antibiotics as a feed
additive in poultry production. Further, the carriage of
antibiotic resistant enteric bacteria by flies in the poultry
production environment increases the potential for human
exposure to drug resistant bacteria.
Acknowledgements
Support for this research was received from the Center for a
Livable Future at the Johns Hopkins Bloomberg School of
Public Health. We would also like to thank Dr. Macovei, Dr.
Jensen, Dr. McDermott, and Patti Cullen for providing control
strains used in our analysis.
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