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Antimicrobial Agents and Chemotherapy, October 2000, p. 2897-2899, Vol. 44, No. 10
Department of Veterinary and Microbiological
Sciences, North Dakota State University, Fargo, North
Dakota1; Division of Immunity and
Infection, University of Birmingham, Birmingham, United
Kingdom3; Department of Avian Medicine,
University of Georgia, Athens, Georgia4; and
Division of Animal and Food Microbiology, Center for Veterinary
Medicine, U.S. Food and Drug Administration, Laurel,
Maryland2
Received 6 April 2000/Returned for modification 21 June
2000/Accepted 10 July 2000
Fluoroquinolone-resistant avian Escherichia coli
isolates from northern Georgia were investigated for gyrA
and parC mutations. All isolates contained a mutation in
GyrA replacing Ser83 with Leu; seven isolates also contained mutations
replacing Asp87 with either Gly or Tyr. Random amplified polymorphic
DNA analysis revealed that quinolone-resistant E. coli
isolates were genetically diverse.
Colibacillosis continues to
significantly contribute to increased mortality and economic losses in
the poultry industry (1, 4, 6, 11). Sarafloxacin and
enrofloxacin were approved in 1995 and 1996 in the United States for
veterinary use to help control morbidity and mortality associated with
Escherichia coli-related colibacillosis infections
(14).
Quinolone resistance mechanisms employed by gram-negative bacteria
include chromosomal mutations that reduce membrane permeability and
decrease drug accumulation or alter DNA topoisomerases (9, 12, 17,
22, 23, 25). Clinical resistance to fluoroquinolones in E. coli, however, is mostly associated with mutations that result in
amino acid changes in the A subunit (gyrA) and the B subunit
(gyrB) of the DNA gyrase and in the parC-encoded
subunit of topoisomerase IV (5, 8, 17, 18, 20, 23, 25).
This study was undertaken to investigate the genetic mechanisms
involved in the emergence of bacterial fluoroquinolone resistance among
pathogenic avian E. coli isolates. Furthermore, isolates were genetically characterized via random amplified polymorphic DNA
(RAPD) analysis to determine if fluoroquinolone resistance was
associated with specific E. coli clones. Fluoroquinolone
resistance was surveyed among avian E. coli organisms
isolated at the Poultry Diagnostic and Research Center (PDRC),
University of Georgia, during a 37-month period from May 1996 to June
1999 (Fig. 1). Five hundred thirty-five
E. coli isolates from clinical cases of avian colibacillosis
were identified at the PDRC during this time. The percentage of
sarafloxacin-resistant avian E. coli isolates steadily
increased from 15% in 1996 to 40% in 1999 (Fig. 1). Dual resistance
to sarafloxacin and enrofloxacin increased from 9% in 1997 to 30% in
1999.
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Characterization of Fluoroquinolone Resistance
among Veterinary Isolates of Avian Escherichia
coli
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FIG. 1.
Emergence of fluoroquinolone resistance among clinical
E. coli isolates in northern Georgia. Five-hundred
thirty-five pathogenic avian E. coli isolates implicated in
colibacillosis were submitted to the PDRC diagnostic laboratory during
a 37-month period from May 1996 to June 1999. Forty-one isolates were
submitted from May to December 1996, 189 isolates in 1997, 228 isolates
in 1998, and 77 isolates from January to June 1999. Sarafloxacin and
enrofloxacin susceptibilities were determined according to NCCLS
standards (16). SRF, sarafloxacin; ENR, enrofloxacin.
Antimicrobial susceptibilities of 29 nalidixic acid-resistant avian
E. coli isolates were determined with agar dilution and broth microdilution methods and interpreted according to the
National Committee for Clinical Laboratory Standards (NCCLS)
guidelines (15, 16). The majority of E. coli isolates were also resistant to several other antimicrobials
tested, particularly sulfamethoxazole (n = 27),
tetracycline (n = 25), streptomycin (n = 24), gentamicin (n = 18), and ampicillin
(n = 16). Resistance to the cephalosporins cephalothin
(n = 6) and ceftiofur (n = 4) was
observed as well. Additionally, nine E. coli isolates were
resistant to chloramphenicol. Sixty-six percent (19 of 29) of
quinolone-resistant E. coli isolates exhibited multiple
resistance to five or more antimicrobials. MICs of nalidixic acid,
enrofloxacin, sarafloxacin, and ciprofloxacin were then determined
using an agar plate dilution method (Table 1). The MIC of the antibiotic was defined
as the concentration (in micrograms per milliliter of agar) at which no
more than two colonies were detected. Ninety-three percent (27 of 29)
of the isolates required >256 µg of nalidixic acid/ml for
inhibition, whereas two isolates required 64 µg/ml (resistant MIC
breakpoint, 
32 µg/ml). Three isolates required 32 µg of
enrofloxacin/ml (resistant MIC breakpoint, 2 µg/ml) and 32 µg of
sarafloxacin/ml (resistant MIC breakpoint, 0.25 µg/ml) for
inhibition, and 13 isolates required 8 µg of sarafloxacin/ml or more
for inhibition. Six isolates displayed intermediate susceptibility to
ciprofloxacin (MIC = 2 µg/ml), and one isolate was
cross-resistant to ciprofloxacin (MIC = 16 µg/ml; resistant MIC
breakpoint, 4 µg/ml) as well as to enrofloxacin (32 µg/ml) and
sarafloxacin (32 µg/ml).
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Single-stranded conformational polymorphism PCR was employed to
investigate the presence of mutations in the quinolone
resistance-determining regions (QRDR) of the gyrA gene of
DNA gyrase and the parC gene of topoisomerase IV as
previously described (8). DNA sequencing of the
gyrA and parC regions using previously described
primers (8) confirmed the initial single-stranded
conformation polymorphism analysis and interpretations (Table 1). All
29 nalidixic acid-resistant E. coli isolates contained the
amino acid substitution Ser83 
Leu in the GyrA QRDR (Table 1).
However, many of these E. coli isolates displayed variable
fluoroquinolone susceptibility patterns. Seven isolates had the
additional amino acid substitution Asp87 Tyr (n = 2) or Asp87 Gly (n = 5) within the GyrA QRDR.
Twenty-three of 29 quinolone-resistant E. coli isolates
assayed contained a silent mutation at Ser85 (AGCAGT) in
parC. However, no mutations conferring amino acid
substitutions were detected in the QRDR of parC among the
quinolone-resistant avian E. coli isolates (Table 1).
Fluoroquinolone-resistant avian E. coli isolates have also been previously identified in Saudi Arabia and Spain (3, 4). However, neither study identified the specific mutations associated with the fluoroquinolone resistance phenotypes. Additionally, Everett et al. observed that the majority of veterinary E. coli isolates (six of eight) resistant to fluoroquinolones isolated in the United Kingdom had mutations only in the QRDR of the gyrA gene (8). The present study suggests that avian E. coli isolates recovered from diseased poultry in the United States display similar resistance phenotypes as well as sharing common resistance mechanisms with those isolates previously described by other European investigators. However, we cannot exclude additional mechanisms that have yet to be identified which may contribute to fluoroquinolone resistance, especially in isolates with high-level resistance.
Avian E. coli isolates were further typed by RAPD using
previously published primers (13). Grouping of E. coli isolates into each cluster or branch in the dendrogram
correlated with similarities in their RAPD DNA pattern (Fig.
2). A total of 16 different clusters or
RAPD types (RT) were identified from 184 avian E. coli
isolates that have been previously described (13). The 29 fluoroquinolone-resistant avian E. coli isolates were typed by RAPD and could be assigned to six different RT groups: RT1, RT5,
RT6, RT7, RT8, and RT10 (Fig. 2). The random association of
fluoroquinolone-resistant avian E. coli isolates among
multiple RTs indicates a genetically diverse population and suggests
that fluoroquinolone resistance has appeared among independent
chromosomal backgrounds and is not due to the emergence of particular
resistant clonal genotypes.
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This study reports the first occurrence of fluoroquinolone resistance in veterinary E. coli isolates recovered from clinical cases of avian colibacillosis in the United States. A marked increase in sarafloxacin and enrofloxacin resistance was observed among pathogenic avian E. coli in northern Georgia from 1996 to 1999. This trend coincides with the approval of these fluoroquinolones in 1995 and 1996 for treatment of E. coli-related poultry infections. Similar results have been seen among other pathogenic E. coli strains and most likely reflect the selection of antibiotic-resistant populations due to therapeutic use of antimicrobials (3, 4, 6, 7, 20, 24). There is mounting evidence that antimicrobial use in veterinary medicine may select for antimicrobial-resistant zoonotic bacterial pathogens (e.g., Salmonella and Campylobacter) (2, 7, 10, 19, 21). This has led to increased pressure to limit fluoroquinolones in animals to preserve the value of these drugs in the treatment of human infections (2, 10, 19, 21). However, the proposed linkage between fluoroquinolone use in agriculture and the occurrence of resistant human enteric bacterial pathogens is still being debated (19). Regardless, the detection of fluoroquinolone bacterial resistance in a veterinary situation stresses the need for the judicious use of these antimicrobials.
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ACKNOWLEDGMENTS |
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We thank Marie Maier and Julie Sherwood for their technical assistance.
This work was supported by grants from the U.S. Poultry and Egg Association (to J.M., D.W., and L.J.V.P.) and USDA-NRICGP grant 9902829 (to D.W., J.M., and S.Z.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD 20708. Phone: (301) 827-8037. Fax: (301) 827-8127. E-mail: dwhite{at}cvm.fda.gov.
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Antimicrobial Agents and Chemotherapy, October 2000, p. 2897-2899, Vol. 44, No. 10
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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