Use of Streptogramin Growth Promoters in Poultry and Isolation of

Use of Streptogramin Growth Promoters in Poultry and Isolation of

MAJOR ARTICLE
Use of Streptogramin Growth Promoters
in Poultry and Isolation of Streptogramin-Resistant
Enterococcus faecium from Humans
Amy L. Kieke,1 Mark A. Borchardt,1 Burney A. Kieke,1 Susan K. Spencer,1 Mary F. Vandermause,1 Kirk E. Smith,2
Selina L. Jawahir,2 and Edward A. Belongia,1 for the Marshfield Enterococcal Study Groupa
1
Marshfield Clinic Research Foundation, Marshfield, Wisconsin; 2Minnesota Department of Health, St. Paul
(See the editorial commentary by Frimodt-Møller and Hammerum, on pages 1191–3, and the article by Jalava et al.,
on pages 1209–16.)
Background. Virginiamycin use in poultry selects for Enterococcus faecium with cross-resistance to quinupristindalfopristin, a drug for vancomycin-resistant E. faecium in humans. We conducted an epidemiologic study of
poultry exposures as risk factors for human carriage of quinupristin-dalfopristin–resistant E. faecium.
Methods. Rectal or fecal samples for E. faecium testing were obtained from 567 newly admitted hospital patients
and 100 healthy vegetarians. Participants were interviewed regarding poultry exposure. Retail poultry washes (160
conventional and 26 antibiotic free) were also tested for the presence of E. faecium. Constitutive and inducible
quinupristin-dalfopristin resistance were assessed in E. faecium isolates, and resistance genes were identified by
polymerase chain reaction.
Results. E. faecium was isolated from 105 patients, 65 vegetarians, and 77 conventional and 23 antibiotic-free
poultry washes. Constitutive quinupristin-dalfopristin resistance was absent in human E. faecium, but 56% of
conventional poultry isolates were quinupristin-dalfopristin resistant. Inducible quinupristin-dalfopristin resistance
was more common in samples from patients than in those from vegetarians and in washes of conventional than
antibiotic-free poultry. Higher poultry consumption was associated with inducible quinupristin-dalfopristin resistance. vatE was present in 38% of E. faecium isolates from patients and none from vegetarians. Touching raw
poultry was associated with the presence of vatE.
Conclusions. Poultry exposure is associated with a quinupristin-dalfopristin resistance gene and inducible
quinupristin-dalfopristin resistance in human fecal E. faecium. The continued use of virginiamycin may increase
the potential for streptogramin-resistant E. faecium infection in humans.
Nosocomial enterococcal infections have become a major cause of sepsis and wound infections in recent years,
and the prevalence of vancomycin and ampicillin resistance in Enterococcus faecium has increased dramatically in hospital populations [1, 2]. The semisynthetic
Received 18 January 2006; accepted 18 April 2006; electronically published 27
September 2006.
Presented in part: 45th Annual Interscience Conference on Antimicrobial Agents
and Chemotherapy, Washington, DC, 16–19 December 2005 (poster C2-256).
Potential conflicts of interest: none reported.
Financial support: US Centers for Disease Control and Prevention (grant RS1/
CCR520634). The findings and conclusions in this article are those of the authors
and do not necessarily represent the views of the funding agency.
a
Study group members are listed after the text.
Reprints or correspondence: Dr. Edward A. Belongia, Epidemiology Research
Center (ML2), Marshfield Clinic Research Foundation, 1000 North Oak Ave., Marshfield, WI 54449 ([email protected]).
The Journal of Infectious Diseases 2006; 194:1200–8
2006 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2006/19409-0004$15.00
1200 • JID 2006:194 (1 November) • Kieke et al.
antimicrobial quinupristin-dalfopristin is 1 of 3 drugs
licensed by the US Food and Drug Administration
(FDA) for the treatment of serious vancomycin-resistant E. faecium infections in the United States. A related
streptogramin antibiotic, virginiamycin, has been used
as a growth promoter in US livestock since 1975. Virginiamycin is administered to poultry in feed to increase weight gain [3]. The drug is also used as a growth
promoter in swine. Several studies have demonstrated
a relationship between the prevalence of quinupristindalfopristin–resistant E. faecium in food animals and
the magnitude of virginiamycin use as a growth promoter [4–6]. Denmark banned virginiamycin use as a
growth promoter in 1997, and the European Union
subsequently banned virginiamycin and 3 other antimicrobial growth promoters [7].
Quinupristin-dalfopristin is a mixture of 2 streptogramin compounds (A and B) that inhibit bacterial
protein synthesis through different mechanisms. Clinical quinupristin-dalfopristin resistance was reported in a small number
of patients during prelicensure studies [8]. In E. faecium, streptogramin A resistance is encoded by the acetyltransferase genes
vatD and vatE, which are located on plasmids [9–11]. Streptogramin B resistance is mediated by erythromycin resistance
methylase (ermB) genes [12, 13]. These genes do not account
for all quinupristin-dalfopristin–resistant E. faecium, and other
mechanisms of resistance remain undefined [14–16].
Microbiological studies have demonstrated that quinupristin-dalfopristin–resistant E. faecium are prevalent on poultry
farms and in retail poultry in the United States [16–19]. Other
studies have shown that feeding virginiamycin to chickens and
turkeys leads to the emergence of quinupristin-dalfopristin–
resistant E. faecium [20–22]. However, the testing of selected
human fecal samples has suggested that human colonization
with quinupristin-dalfopristin–resistant E. faecium is rare in the
United States [19]. The latter observation was a key factor in
a proposed FDA risk-assessment model regarding streptogramin
use in animals and human infections with quinupristin-dalfopristin–resistant E. faecium. We conducted an epidemiologic
study of quinupristin-dalfopristin susceptibility in E. faecium
colonizing humans and retail poultry and assessed the association between poultry exposure and the isolation of quinupristin-dalfopristin–resistant E. faecium from the gastrointestinal tract of humans.
PATIENTS, MATERIALS, AND METHODS
Study design and population. This was a cross-sectional survey of quinupristin-dalfopristin–resistant E. faecium in recently
admitted hospital patients, healthy vegetarians, and retail poultry in 4 communities. The enrollment community hospitals
included St. Joseph’s Hospital, Marshfield, Wisconsin; Gundersen Lutheran Hospital, La Crosse, Wisconsin; Sacred Heart
Hospital, Eau Claire, Wisconsin; and Rice Memorial Hospital,
Willmar, Minnesota.
Participant recruitment. Newly admitted hospital patients
were enrolled from June 2002 through May 2003. Subjects were
enrolled to yield ∼150 participants per hospital, distributed
evenly throughout the 12-month period. Patients ⭓14 years
old admitted to a medical, surgical, or intensive care unit (ICU)
bed were eligible if they were alert, stable, and capable of giving
informed consent. Patients with acute rectal bleeding, recent
use of cathartics, or neutropenia were excluded. Rectal-swab
samples were obtained within 36 h of hospital admission for
all patients.
Vegetarian participants were recruited from January through
July 2004 at health-food cooperatives located in or near the 4
study communities. Adult vegetarians were eligible to participate if they reported no consumption of meat products for at
least 1 year. Stool samples were obtained from vegetarian participants for the isolation and characterization of E. faecium.
Hospital patients and vegetarians were interviewed by telephone regarding potential dietary and environmental sources
of E. faecium. Interviews were conducted within 62 days of
enrollment for hospital patients and at the time of enrollment
for vegetarians. The main exposures of interest included touching raw meat, consuming meat products, and contact with live
food animals. For hospital patients, information on recent antibiotic use and chronic diseases was obtained through abstraction of medical records. The study protocol was approved by
the Marshfield Clinic’s institutional review board, and all participants provided written, informed consent.
Retail poultry samples. Retail poultry samples were collected 4 times during the 12-month hospital recruitment period
from grocery stores in the 4 study communities. During each
visit to a community, we purchased a convenience sample of
∼8 chicken and 2 turkey products from various retail locations.
These retail samples, designated “conventional retail poultry,”
were selected without regard to label information concerning
antibiotic use on the farm. During an additional collection
period, retail poultry products were purchased from food cooperatives and natural-food stores in or near the study communities. These poultry products, designated “antibiotic-free
retail poultry,” were confirmed to be raised without antibiotics
on the basis of the product label or by contacting the manufacturer or distributor.
Laboratory methods. Stool samples and rectal swabs were
suspended in brain-heart infusion (BHI) broth overnight at
37C. Retail poultry samples were massaged in buffered peptone
water, and the rinsate was centrifuged at 3070 g to yield 1 mL
of concentrate for inoculation into BHI. E. faecium was isolated
on colistin–naladixic acid agar, identified using standard biochemical procedures, and confirmed by polymerase chain reaction (PCR) for a species-specific ddl amplicon [23]. A total
of 5–6 randomly selected isolates from each sample were evaluated for resistance to quinupristin-dalfopristin. Quinupristindalfopristin susceptibility was measured by E-test using a recognized reference standard for the interpretation of MICs [24].
Isolates with MICs ⭓4 mg/mL were classified as resistant, and
those with MICs between 1 and 4 mg/mL were classified as
having intermediate resistance.
An E. faecium isolate representing each level of quinupristindalfopristin resistance (susceptible, intermediate, or resistant)
found in each sample (5–6 isolates) was selected for further
evaluation. Ten genetic markers were evaluated initially in the
hospital patients: vatA, vatB, vatC, vgaA, vgaB, vgbA, vgbB, vatE,
vatD, and ermB [13–16, 25, 26]. Only the latter 3 were identified,
and subsequent PCR testing for genetic markers was limited to
this group. Positive results were confirmed by DNA sequencing.
Representative isolates from humans and retail poultry were charStreptogramin-Resistant E. faecium • JID 2006:194 (1 November) • 1201
Table 1. Characteristics of newly admitted hospital patients and healthy vegetarians colonized with Enterococcus faecium.
Hospital patients
(n p 105)
Vegetarians
(n p 65)
Age, mean SD, years
Male sex
63 17
62 (59)
39 15
23 (35)
White race
College graduate
Employed
⭓5 physician visits during the preceding year
104
21
46
56
Characteristic
(99)
(20)
(44)
(53)
63
42
47
7
(97)
(65)
(72)
(11)
Exposures during the month before enrollment
a
Antibiotic therapy
Exposed to live poultry
60 (57)
9 (9)
0
15 (23)
Touched raw poultry
Poultry consumption
54 (53)
98 (96)
6 (9)
0
9
0
No. of times poultry consumed during the
preceding month, median
NOTE. Data are no. (%), unless otherwise indicated. Missing values for hospital patients
included: 1 for college graduate, 2 for exposure to live poultry, 3 for touching raw poultry, 3 for
poultry consumption, and 6 for frequency of poultry consumption. One vegetarian had missing
information for touching raw poultry.
a
Antibiotic therapy was based on self-report for vegetarians and on medical record review
for hospital patients.
acterized using pulsed-field gel electrophoresis (PFGE) at the
Minnesota Department of Health (St. Paul) [27].
E. faecium isolates that were sensitive or had intermediate
resistance to quinupristin-dalfopristin (MICs !4 mg/mL) were
tested for the detection of inducible resistance [28]. Initially,
5 ⫻ 10 6 E. faecium (measured by optical density) in the log
phase of growth were transferred to 5 mL of BHI broth that
contained 0.25 mg/mL virginiamycin and cultured at 37C for
24 h. The bacterial density was readjusted to 1 ⫻ 10 6/mL in
fresh BHI broth, challenged with 8 mg/mL quinupristin-dalfopristin, and incubated for another 24 h at 37C. The level of
inducible quinupristin-dalfopristin resistance was expressed as
relative growth, defined as the 24-h optical density (at 600 nm)
of the isolate preexposed to virginiamycin and challenged with
quinupristin-dalfopristin divided by the optical density of the
same isolate cultured in BHI broth for 24 h without virginiamycin before exposure or quinupristin-dalfopristin challenge.
This ratio was multiplied by 100 to yield the relative percentage
of growth, with possible values ranging from 0% (no inducible
resistance) to 100% (high level of inducible resistance). The
inducibility assay was also performed for isolates from hospital
patients after preexposure to 0.25 mg/mL of quinupristin-dalfopristin instead of virginiamycin.
Statistical analysis. Statistical analyses were based on the
E. faecium isolate with the highest level of constitutive quinupristin-dalfopristin resistance in each sample. The primary
outcomes included the presence of the vatE gene and the level
of inducible resistance, measured as the relative percentage of
growth. Contact with raw poultry and frequency of poultry
1202 • JID 2006:194 (1 November) • Kieke et al.
consumption were the primary exposures of interest. The latter
was dichotomized at the median consumption level for hospital
patients. Vegetarians made up a separate category of poultry
consumption.
Unadjusted associations between the exposure and outcome
measures were assessed using Fisher’s exact test, the Wilcoxon
rank-sum test, or the Kruskal-Wallis test. Multivariable regression models were used to estimate adjusted measures of association. Separate models were constructed for each combination of population (hospital patients alone or the combined
group of hospital patients and vegetarians), outcome, and exposure. Variables were screened for inclusion in an initial multivariable model. Candidate variables with P ⭐ 0.20 were retained. Collinearity was assessed in the resulting model [29,
30]. Final models included variables that achieved statistical
significance (P ⭐ 0.05) or whose exclusion altered the outcomeexposure measure of association by 110%.
For the vatE models, adjusted prevalence ratios were computed using methods described by Zou [31]. Linear regression
was used for the inducible resistance models. Because the distribution of inducible resistance was skewed, the natural log
transformation was applied before models were fitted. Applying
the inverse transformation to the fitted linear model resulted
in a model of the median response on the original scale [32].
The measure of association for the inducible resistance models
was the median relative percentage of growth in the exposed
group divided by that in the unexposed group. For both measures of association (prevalence ratios for vatE and ratios of
medians for inducible resistance), a value of 1.0 indicated no
Table 2. Quinupristin-dalfopristin susceptibility (E-test) and genetic determinants of resistance in Enterococcus faecium from different ecologic sources.
Susceptibility to quinupristin-dalfopristin
E. faecium source
Hospital patients (n p 105)
Vegetarians (n p 65)
Conventional retail poultry (n p 77)
Antibiotic-free retail poultrya (n p 23)
Resistance genes
Susceptible
Intermediate
Resistant
vatE
ermB
25 (24)
8 (12)
11 (14)
80 (76)
57 (88)
23 (30)
0
0
43 (56)
40 (38)
0
36 (47)
9 (9)
0
31 (40)
11 (48)
9 (39)
3 (13)
3 (13)
2 (9)
NOTE. Data are no. (%) of isolates.
a
Antibiotic-free retail poultry samples were obtained from food cooperatives or natural food stores and were confirmed to
be raised without antibiotics by product label or information provided by the producer or distributor.
effect, and values 11.0 indicated an association between the
exposure and outcome of interest. All analyses were performed
using SAS software (version 9.1; SAS Institute).
The following variables were screened for inclusion in the
multivariable models: touching raw poultry; touching raw beef;
touching raw pork; frequency of consumption of poultry, beef,
and pork; cooking one’s own meals; age; sex; education; employment status; state of residence; alcohol consumption; and
physician visits, hospitalizations, and ICU admissions during
the prior year. Vegetarian status was screened for inclusion in
the models when relevant. For analyses of hospital patients
alone, hospital location and a comorbidity index [33] were also
assessed for inclusion in the models.
RESULTS
There were 1614 eligible patients contacted within 36 h after
hospital admission, and 622 (39%) agreed to participate. Of
these, 55 were excluded from the analysis because of specimen
shipping delays (n p 12) or because the risk-factor interview
was not completed (n p 43). For the remaining 567 patients,
the median interval from hospital admission to fecal specimen
collection was 25 h (range, 11–36 h). The median time between
enrollment and completion of the risk-factor interview was 22
days (range, 5–62 days). One hundred vegetarians provided
stool samples and completed the risk-factor interview. E. faecium was isolated from 105 hospital patients and 65 vegetarians;
further analyses were restricted to this group. Compared with
vegetarians, the hospital patients were more likely to be older
and male, with less formal education (table 1).
None of the human E. faecium isolates had constitutive resistance to quinupristin-dalfopristin (MIC ⭓4 mg/mL) by Etest, but a majority of isolates from both hospital patients and
vegetarians had intermediate quinupristin-dalfopristin resistance (table 2). Neither vatE nor ermB was found in vegetarian
E. faecium isolates; vatE was commonly found in isolates from
the hospital patients. ErmB was present in !10% of hospital
patient isolates, and vatD was found in 1 E. faecium isolate
from a hospital patient. The rarity of ermB and vatD genes in
the study population prevented the effective evaluation of their
role in the association between poultry exposure and quinupristin-dalfopristin resistance.
E. faecium was isolated from 77 (48%) of 160 conventional
retail poultry samples and 23 (88%) of 26 antibiotic-free retail
poultry samples. Quinupristin-dalfopristin resistance was more
common in isolates from conventional than antibiotic-free retail poultry (table 2). Conventional retail poultry isolates were
∼4 times more likely to contain vatE (P p .004) and ermB
(P p .005) than were isolates from antibiotic-free retail poultry.
All PFGE patterns from humans and retail poultry were distinct,
and no common clones were identified in both sources (data
not shown).
The level of inducible resistance to quinupristin-dalfopristin
(expressed as relative percentage of growth) varied greatly in
E. faecium isolates from different sources (figure 1). Higher
inducible resistance levels were found in E. faecium isolates from
hospital patients than in those from vegetarians. The median
relative percentage of growth was 6.5% (range, 1.3%–56.4%)
for isolates from hospital patients and 2.4% (range, 1.2%–4.6%)
for isolates from vegetarians (P ! .001 ). For isolates from hospital patients that were preexposed to quinupristin-dalfopristin,
the median relative percentage of growth was 6.1% (range,
0.8%–45.8%). Without preexposure to virginiamycin or quinupristin-dalfopristin, isolates from hospital patients did not
grow when challenged with 8 mg/mL quinupristin-dalfopristin
(median relative percentage of growth, 2.0%; range, 0.9%–
8.2%). When measured by E-test, 25 (24%) of 105 isolates from
hospital patients became resistant to quinupristin-dalfopristin
(MIC ⭓4 mg/mL) after preexposure to virginiamycin; the median MIC of these resistant isolates after induction was 8 mg/
mL (range, 4–32 mg/mL). The relative percentage of growth
after preexposure to virginiamycin and quinupristin-dalfopristin challenge was correlated with the MIC measurement of
resistance after induction (Spearman’s correlation, 0.55 [95%
confidence interval {CI}, 0.40–0.67]).
Inducible quinupristin-dalfopristin resistance was associated
with the presence of vatE. The median relative percentage of
Streptogramin-Resistant E. faecium • JID 2006:194 (1 November) • 1203
Figure 1. Plots of inducible resistance for Enterococcus faecium isolated from humans and retail poultry with initial E-test MICs !4 mg/mL for
quinupristin-dalfopristin. Each symbol represents 1 isolate. Inducible resistance (relative percentage of growth) was determined by dividing the 24-h
growth density of isolates after preexposure to virginiamycin and quinupristin-dalfopristin challenge by the 24-h growth density of untreated isolates
and multiplying by 100.
growth after preexposure to virginiamycin was 15.8% (interquartile range [IQR], 9.0%–26.2%) for isolates that contained
vatE and 2.6% (IQR, 2.0%–3.7%) for those without vatE
(P ! .001). The PFGE patterns of the inducible isolates were
distinct from each other. For each isolate, the pattern after
induction and quinupristin-dalfopristin challenge was indistinguishable from the original pattern before induction, which is
consistent with inducible resistance rather than selection of a
contaminating isolate with constitutive resistance.
E. faecium isolates from conventional retail poultry had
higher levels of inducible resistance than isolates from antibiotic-free retail poultry. The median relative percentage of
growth was 30.6% (range, 1.4%–47.8%) for conventional retail
poultry isolates and 2.2% (range, 1.5%–6.6%) for antibioticfree retail poultry isolates (P ! .001).
Risk-factor analysis. The association between poultry exposures and carriage of E. faecium with vatE differed among
hospital patients with and without prior antibiotic use. Fortyfive (43%) of 105 hospital patients with E. faecium had used
antibiotics during the month before enrollment. The most common antibiotics used were b-lactams (70%), fluoroquinolones
(29%), and macrolides (9%). Among these patients, touching
1204 • JID 2006:194 (1 November) • Kieke et al.
raw poultry did not increase the risk of carrying E. faecium
with vatE (prevalence ratio, 0.55 [95% CI, 0.31–0.96]). By contrast, the hospital patients without recent antibiotic use had a
increased risk of carrying E. faecium isolates with vatE if they
had touched raw poultry (prevalence ratio, 1.94 [95% CI, 0.73–
5.15]; P ⭐ .05 for both exposures, Breslow-Day test) [34]. None
of the vegetarians with E. faecium had used antibiotics during
the month before enrollment. On the basis of these findings,
results are reported for participants without recent antibiotic use.
Carriage of E. faecium with vatE was significantly associated
with both touching raw poultry and higher poultry consumption in the combined hospital patient and vegetarian group
(table 3). Inducible resistance was significantly associated with
higher poultry consumption in this group. In the multivariable
models, we found higher point estimates for both raw poultry
contact and poultry consumption in persons without recent
antibiotic use, but not all of these were statistically significant
(table 4). Carriage of E. faecium with vatE was significantly
associated with touching raw poultry in the combined hospital
patient and vegetarian group (P p .032 ). Because none of the
vegetarian isolates had the vatE gene, we were unable to assess
the association between vatE and poultry consumption in the
Table 3. Unadjusted association between vatE or inducible resistance (relative percentage
of growth) and poultry exposure in Enterococcus faecium from hospital patients and vegetarians.
Group
vatE present
No. with
b
E. faecium
No. (%)
18
7 (39)
25
5 (20)
20
8 (40)
24
P
c
Inducible
a
resistance
Median
P
c
Hospital patients only (n p 45)
Touched raw poultry
Yes
No
Poultry consumptiond
High
Low
Hospital patients and vegetarians (n p 110)
Touched raw poultry
Yes
No
4.6
.30
3.6
.58
4 (17)
.10
3.2
.13
24
83
7 (29)
5 (6)
.005
3.0
2.5
.10
20
24
65
8 (40)
4 (17)
0
7.8
Poultry consumptiond
High
Low
Veg
!.001
7.8
3.2
2.4
!.001
NOTE. Results are shown for participants without antibiotic use during the preceding month.
a
Inducible resistance (relative percentage of growth) was determined by dividing the 24-h growth density of
isolates after preexposure to virginiamycin and quinupristin-dalfopristin challenge by the 24-h growth density of
untreated isolates and multiplying by 100.
b
Two and 1 hospital patients who did not use antibiotics in the past month were missing data on touching raw
poultry and frequency of poultry consumption, respectively. One vegetarian was missing data on touching raw
poultry.
c
Fisher’s exact P for the dichotomous vatE measure. For the continuous inducible resistance (relative percentage
of growth) measure, either the Wilcoxon rank-sum test (dichotomous exposure) or the Kruskal-Wallis test (3category exposure) was used.
d
Poultry consumption was defined as “high” for those who consumed poultry above the median for the hospital
patients (9 times/month), “low” for hospital patients who consumed less, and “veg” for vegetarians.
combined group using statistical methods. However, this association was marginally statistically significant (P p .058 ) in
the hospital patients. Carriage of E. faecium with inducible
resistance (higher relative percentage of growth) was significantly associated with touching raw poultry in the hospital
patients (P p .027). Inducible resistance was also significantly
associated with higher poultry consumption in the combined
group of hospital patients and vegetarians (P ! .001 for both
consumption levels in hospital patients vs. vegetarians).
DISCUSSION
The results of the present investigation suggest that virginiamycin use in poultry contributes to human carriage of E. faecium that contains streptogramin resistance genes with readily
inducible resistance. A previous study in the United States
found that fecal E. faecium with constitutive quinupristin-dalfopristin resistance are rare in humans, but that study did not
evaluate the prevalence of streptogramin resistance genes or
inducible resistance [19]. In the present study, susceptible and
intermediate isolates were screened for both resistance genes
and inducible resistance. The acetyltransferase gene vatE was
commonly found in both human fecal E. faecium and in isolates
colonizing retail poultry. In the risk-factor analysis, touching
raw poultry and more frequent poultry consumption were independently associated with the presence of vatE and a phenotype of inducible quinupristin-dalfopristin resistance. In particular, we found striking differences in the characteristics of
E. faecium from recently hospitalized patients who ate meat,
compared with healthy vegetarians. The vatE gene was not
found in any vegetarian E. faecium isolates, but it was found
in approximately one-third of isolates from hospital patients.
The median level of inducible resistance was also 3-fold higher
in persons with a high level of poultry consumption, compared
with vegetarians. These positive associations with poultry contact and consumption were found only in persons without
recent antibiotic use.
The identification of poultry-related risk factors in the present
study is supported by previous studies that have reported a
high prevalence of quinupristin-dalfopristin–resistant E. faecium in retail poultry in the United States [16–18]. Other investigators have also shown that streptogramin-resistant E. faecium occur less frequently in animals raised without antibiotics
Streptogramin-Resistant E. faecium • JID 2006:194 (1 November) • 1205
Table 4. Adjusted association between vatE or inducible resistance (relative percentage of
growth) and poultry exposure in Enterococcus faecium from hospital patients and vegetarians.
Inducible
resistance
vatE present
Group
Adjusted
prevalence ratio
95% CI
Adjusted
ratioa
95% CI
Hospital patients only
Touched raw poultry
2.3
0.7–6.9
2.1
1.1–4.1
3.0
1.0–9.2
1.5
0.8–2.7
Touched raw poultry
High poultry consumption vs. vegetarianb
3.4
1.1–10.4
1.4
0.9–2.2
…
…
5.7
3.3–9.9
Low poultry consumption vs. vegetarianb
…
…
4.0
2.4–6.5
High poultry consumption
Hospital patients and vegetarians
NOTE. Results are shown for participants without antibiotic use during the preceding month. CI, confidence
interval.
a
Estimated median relative percentage of growth in the exposed group divided by that in the unexposed
group. An adjusted ratio 11 indicates an association between the exposure and relative percentage of growth.
b
Prevalence ratios for vatE were undefined because none of the vegetarians carried E. faecium with vatE.
[20–22], and we found that inducible resistance was substantially lower in antibiotic-free retail poultry isolates, relative to
those in conventional poultry. The biologic plausibility for the
foodborne acquisition of streptogramin resistance genes is further supported by the observation that vatE is located on plasmids and can be transferred by conjugation [16, 35, 36]. In an
experimental study, ingestion of streptogramin-resistant E. faecium led to transient colonization for up to 14 days, which
provides sufficient time for conjugative transfer of resistance
determinants to endogenous flora [37]. In the present study,
PFGE failed to identify similar strains in humans and retail
poultry, which is consistent with the hypothesis that the horizontal transfer of resistance genes is more important than
clonal spread of E. faecium from livestock to humans.
The continued nontherapeutic use of virginiamycin and
other growth promoters in livestock has been controversial.
Virginiamycin use has been banned in Denmark and the European Union, but no regulatory action has been taken by the
FDA to prohibit its use in the United States. The FDA has
classified streptogramin antibiotics as “highly important” for
use in human medicine (available at: http://www.fda.gov/cvm/
guidance/fguide152.pdf), and a risk-assessment model for
streptogramin use in food-producing animals has been under
development (available at: http://www.fda.gov/cvm/Documents/
SREF_RA_FinalDraft.pdf). However, the lack of critical information about the probability of acquiring streptogramin resistance through the food supply and the potential for human
health consequences has made it difficult to develop meaningful
risk estimates. The FDA risk assessment assumed that only 10%
of quinupristin-dalfopristin–resistant E. faecium infections originated from food pathways. The results of the present study
suggest that the FDA model may underestimate the true risk
of foodborne acquisition, because streptogramin resistance
1206 • JID 2006:194 (1 November) • Kieke et al.
genes are commonly found in human fecal E. faecium, despite
the absence of constitutive streptogramin resistance.
The presence of vatE and inducible streptogramin resistance
in the endogenous fecal flora of newly hospitalized patients
creates a genetic reservoir for the emergence of streptograminresistant, vancomycin-resistant E. faecium in the hospital environment. E. faecium genogroups have been shown to be associated with different ecologic sources, and isolates carried by
healthy people are distinct from those causing nosocomial infection [38, 39]. However, other investigators have suggested
that the previous spread of vancomycin-resistant E. faecium infections was due to nosocomial selection of a specific ampicillinresistant E. faecium genotype, with subsequent horizontal transfer
of the vanA transposon [39]. We hypothesize that a similar mechanism of spread could occur with streptogramin-resistant E. faecium, although the genetic basis for streptogramin resistance has
not been fully elucidated. The selection and horizontal transmission of streptogramin-resistant E. faecium may be clinically
unimportant in the present era because of the infrequent use of
quinupristin-dalfopristin in hospitals. However, if the use of
quinupristin-dalfopristin increases in future years, the presence
of vatE and other genes might facilitate the rapid emergence of
streptogramin resistance.
There are several caveats with regard to the design and interpretation of the present study. Healthy vegetarians differ
from hospitalized patients in many characteristics besides poultry consumption. Although the multivariable analysis adjusted
for many of these factors, confounding may have occurred, and
other factors associated with vegetarian status may have contributed to the observed associations. In addition, this was a
cross-sectional study, and the temporal relationship between
poultry exposures and E. faecium characteristics could not be
determined. Finally, the food-consumption patterns and other
exposures were self-reported, and we could not independently
validate the responses. Misclassification of poultry exposures
may have occurred, although we expect that any such misclassification would be nondifferential. Additional prospective
studies in different populations would be helpful in confirming
the major findings. In particular, the relationship between the
presence of streptogramin resistance genes and poultry exposure should be assessed in the general population, where recent
antibiotic exposure is uncommon. It would also be helpful to
conduct a similar study in a country where virginiamycin is
not used as a growth promoter.
These findings raise additional concerns regarding the continued use of virginiamycin in food animals. Therapeutic options for serious human vancomycin-resistant enterococcal infections are limited, and quinupristin-dalfopristin is one of few
drugs that remain effective. With few new antimicrobials under
development, a high priority should be placed on assessing the
human health impact of continued streptogramin use in food
animals and on developing evidence-based policies to prevent
the emergence of streptogramin-resistant infections in hospitals.
5.
6.
7.
8.
9.
10.
MARSHFIELD ENTEROCOCCAL STUDY GROUP
MEMBERS
11.
Members of the Marshfield Enterococcal Study Group include
Debra L. Kempf and Sonia S. Stratman (Marshfield Clinic Research Foundation, Marshfield, WI); Joanne M. Bartkus and
Kristin Pederson Gulrud (Minnesota Department of Health,
Minneapolis); James R. Johnson (Veterans Affairs Medical Center and the University of Minnesota, Minneapolis); and Jeffrey
B. Bender (University of Minnesota College of Veterinary Medicine, St. Paul).
12.
13.
14.
15.
Acknowledgments
16.
We thank William Agger, Phillip Bertz, Carol Beyer, Jeanne Boller, David
Boxrud, Kathleen Brecke, Marilyn Bruger, Meghan Cheyne, Lacy Doleshal,
Theresa Esser, Juanita Herr, Deborah Hilgemann, Helen Jo Horn, Lynn
Ivacic, Jean McKay, Jordon Ott, Bridget Pfaff, Barbara Piasecki, Salah
Qutaishat, Carla Rottscheit, Jacklyn Salzwedel, Joni Scheftel, Judy Simpson,
Sandra Strey, and Donna Wittman, for their contributions to this project;
and Fred Angulo and Todd Weber, for their review of the manuscript.
17.
References
1. Huycke M, Sahm D, Gilmore M. Multiple-drug resistant enterococci:
the nature of the problem and an agenda for the future. Emerg Infect
Dis 1998; 4:239–49.
2. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond
MB. Nosocomial bloodstream infections in US hospitals: analysis of
24,179 cases from a prospective nationwide surveillance study. Clin
Infect Dis 2004; 39:309–17 (erratum Clin Infect Dis 2004; 39:1093).
3. Hershberger E, Donabedian S, Konstantinou K, Zervos MJ. Quinupristin-dalfopristin resistance in gram-positive bacteria: mechanism of
resistance and epidemiology. Clin Infect Dis 2004; 38:92–8.
4. Werner G, Klare I, Witte W. Association between quinupristin/dalfo-
18.
19.
20.
21.
22.
23.
pristin resistance in glycopeptide-resistant Enterococcus faecium and the
use of additives in animal feed. Eur J Clin Microbiol Infect Dis 1998;17:
401–2.
Aarestrup FM, Kruse H, Tast E, Hammerum AM, Jensen LB. Associations between the use of antimicrobial agents for growth promotion
and the occurrence of resistance among Enterococcus faecium from
broilers and pigs in Denmark, Finland, and Norway. Microb Drug
Resist 2000; 6:63–70.
Aarestrup F, Seyfarth A, Emborg H, Pedersen K, Hendriksen R, Bager
F. Effect of abolishment of the use of antimicrobial agents for growth
promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in Denmark. Antimicrob Agents Chemother
2001; 45:2054–9.
Wegener HC. Ending the use of antimicrobial growth promoters is
making a difference. ASM News 2003; 69:443–8.
Moellering RC, Linden PK, Reinhardt J, Blumberg EA, Bompart F,
Talbot GH. The efficacy and safety of quinupristin/dalfopristin for the
treatment of infections caused by vancomycin-resistant Enterococcus
faecium: Synercid Emergency-Use Study Group. J Antimicrob Chemother 1999; 44:251–61.
Werner G, Witte W. Characterization of a new enterococcal gene, satG,
encoding a putative acetyltransferase conferring resistance to streptogramin A compounds. Antimicrob Agents Chemother 1999; 43:1813–4.
Rende-Fournier R, Leclercq R, Galimand M, Duval J, Courvalin P.
Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145. Antimicrob Agents Chemother
1993; 37:2119–25.
Hammerum AM, Jensen LB, Aarestrup FM. Detection of the satA gene
and transferability of virginiamycin resistance in Enterococcus faecium
from food-animals. FEMS Microbiol Lett 1998; 168:145–51.
Weisblum B. Erythromycin resistance by ribosome modification. Antimicrob Agents Chemother 1995; 39:577–85.
Jensen LB, Hammerum AM, Aerestrup FM, van den Bogaard AE,
Stobberingh EE. Occurrence of satA and vgb genes in streptograminresistant Enterococcus faecium isolates of animal and human origins in
the Netherlands [letter]. Antimicrob Agents Chemother 1998; 42:3330–1.
Werner G, Klare I, Heier H, et al. Quinupristin/dalfopristin-resistant
enterococci of the satA (vatD) and satG (vatE) genotypes from different
ecological origins in Germany. Microb Drug Resist 2000; 6:37–47.
Soltani M, Beighton D, Philpott-Howard J, Woodford N. Mechanisms
of resistance to quinupristin-dalfopristin among isolates of Enterococcus
faecium from animals, raw meat, and hospital patients in Western
Europe. Antimicrob Agents Chemother 2000; 44:433–6.
Simjee S, White DG, Meng J, et al. Prevalence of streptogramin resistance genes among Enterococcus isolates recovered from retail meats
in the greater Washington DC area. J Antimicrob Chemother 2002;
50:877–82.
Hayes JR, English LL, Carter PJ, et al. Prevalence and antimicrobial
resistance of Enterococcus species isolated from retail meats. Appl Environ Microbiol 2003; 69:7153–60.
Hayes JR, McIntosh AC, Qaiyumi S, et al. High-frequency recovery of
quinupristin-dalfopristin-resistant Enterococcus faecium isolates from
the poultry production environment. J Clin Microbiol 2001; 39:2298–9.
McDonald LC, Rossiter S, Mackinson C, et al. Quinupristin-dalfopristin-resistant Enterococcus faecium on chicken and in human stool
specimens. N Engl J Med 2001; 345:1155–60.
Welton LA, Thal LA, Perri MB, et al. Antimicrobial resistance in enterococci isolated from turkey flocks fed virginiamycin. Antimicrob
Agents Chemother 1998; 42:705–8.
Donabedian S, Thal LA, Bozigar P, Zervos T, Hershberger E, Zervos
M. Antimicrobial resistance in swine and chickens fed virginiamycin
for growth promotion. J Microbiol Methods 2003; 55:739–43.
Hershberger E, Oprea SF, Donabedian SM, et al. Epidemiology of
antimicrobial resistance in enterococci of animal origin. J Antimicrob
Chemother 2005; 55:127–30.
Dutka-Malen S, Evers A, Courvalin P. Detection of glycopeptide re-
Streptogramin-Resistant E. faecium • JID 2006:194 (1 November) • 1207
24.
25.
26.
27.
28.
29.
30.
sistance genotypes and identification to the species level of clinically
relevant enterococci by PCR. J Clin Microbiol 1995; 33:24–7.
NCCLS. Methods for determining bactericidal activity of antimicrobial
agents. Wayne, PA: NCCLS, 1999.
Haroche J, Allignet J, Buchrieser C, El Solh N. Characterization of a
variant of vga(A) conferring resistance to streptogramin A and related
compounds. Antimicrob Agents Chemother 2000; 44:2271–5.
Bozdogan B, Leclercq R. Effects of genes encoding resistance to streptogramins A and B on the activity of quinupristin-dalfopristin against
Enterococcus faecium. Antimicrob Agents Chemother 1999; 43:2720–5.
Duck WM, Steward CD, Banerjee SN, McGowan JE Jr, Tenover FC.
Optimization of computer software settings improves accuracy of
pulsed-field gel electrophoresis macrorestriction fragment pattern analysis. J Clin Microbiol 2003; 41:3035–42.
Borchardt MA, Spencer S, Vandermause M, Kieke A, Kieke B, Belongia
E. Quinupristin-dalfopristin resistance in human Enterococcus faecium
increases with pre-exposure to virginiamycin [abstract C2-1991). In:
Program and abstracts of the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy (Washington, DC). Washington,
DC: American Society for Microbiology, 2004:131.
Belsley DA, Kuh E, Welsch RE. Regression diagnostics. New York: John
Wiley, 1980.
Davis CE, Hyde JE, Bangdiwala SI, Nelson JJ. An example of dependencies among variables in a conditional logistic regression. In: Mool-
1208 • JID 2006:194 (1 November) • Kieke et al.
31.
32.
33.
34.
35.
36.
37.
38.
39.
gavkar SH, Prentice RL, eds. Modern statistical methods in chronic
disease epidemiology. New York: John Wiley, 1986:140–7.
Zou G. A modified Poisson regression approach to prospective studies
with binary data. Am J Epidemiol 2004; 159:702–6.
Miller DM. Reducing transformation bias in curve fitting. Am Stat
1984; 38:124–6.
Charlson M, Pompei P, Ales K, MacKenzie C. A new method of classifying prognostic comorbidity in longitudinal studies: development
and validation. J Chron Dis 1987; 40:373–83.
Agresti A. An introduction to categorical data analysis. New York: John
Wiley & Sons, 1996.
Simjee S, White DG, Wagner DD, et al. Identification of vat(E) in
Enterococcus faecalis isolates from retail poultry and its transferability to
Enterococcus faecium. Antimicrob Agents Chemother 2002; 46:3823–8.
Werner G, Klare I, Witte W. Molecular analysis of streptogramin resistance in enterococci. Int J Med Microbiol 2002; 292:81–94.
Sorensen TL, Blom M, Monnet DL, Frimodt-Moller N, Poulsen RL,
Espersen F. Transient intestinal carriage after ingestion of antibioticresistant Enterococcus faecium from chicken and pork. N Engl J Med
2001; 345:1161–6.
Willems RJ, Top J, van den Braak N, et al. Molecular diversity and
evolutionary relationships of Tn1546-like elements in enterococci from
humans and animals. Antimicrob Agents Chemother 1999; 43:483–91.
Leavis HL, Willems RJ, Top J, et al. Epidemic and nonepidemic multidrug-resistant Enterococcus faecium. Emerg Infect Dis 2003; 9:1108–15.