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Antimicrobial Agents and Chemotherapy, February 2000, p. 421-424, Vol. 44, No. 2
Department of Microbiology and
Parasitology1 and Department of Avian
Medicine,2 College of Veterinary Medicine, The
University of Georgia, Athens, Georgia 30602, and Center for
Veterinary Medicine, Food and Drug Administration, Laurel, Maryland
207083
Received 21 July 1999/Returned for modification 17 September
1999/Accepted 3 November 1999
Florfenicol is an antibiotic approved for veterinary use in cattle
in the United States in 1996. Although this drug is not used in
poultry, we have detected resistance to florfenicol in clinical
isolates of avian Escherichia coli. Molecular typing demonstrated that the florfenicol resistance gene, flo, was
independently acquired and is plasmid encoded.
Florfenicol is a synthetic,
fluorinated analogue of chloramphenicol which lacks chloramphenicol's
associated human health risk (11). It has been used in Asia
for aquaculture since the 1980's (12). In early 1996, an
injectable formulation of florfenicol was approved for the treatment of
bovine respiratory disease in the United States. It has not yet been
approved for poultry, and, in fact, an animal feed formulation is not available.
Florfenicol is bacteriostatic, and its mechanism of action is similar
to that of chloramphenicol (7, 22). The mechanism of
resistance to florfenicol is unknown but is associated with the
flo determinant, a highly conserved gene sequence detected in Salmonella enterica serovar Typhimurium DT104 (4,
6) and in the fish pathogen Pasteurella piscicida
(Photobacterium damsela) (15). The flo
gene confers resistance to both chloramphenicol and florfenicol
(4, 14).
Resistance to chloramphenicol is most commonly mediated by mono- and
diacetylation via chloramphenicol acetyltransferase (CAT) enzymes,
which prevents the binding of chloramphenicol to the 50S ribosomal
subunit (21). None of the genes encoding CAT has been shown
to confer resistance to florfenicol, and there is no homology between
the CATs and Flo (9). Another mediator of chloramphenicol
resistance, the cmlA gene of Pseudomonas
aeruginosa, is believed to be a nonenzymatic efflux pump
(3). CmlA is approximately 50% similar in amino acid
sequence to Flo (4), but it is not known whether
cmlA confers resistance to florfenicol.
Our study examined the prevalence of florfenicol resistance in clinical
avian Escherichia coli isolates. We hypothesized that there
were preexisting genes in these bacterial isolates that conferred
resistance to florfenicol and that this resistance might limit the
future usefulness of the drug in other veterinary species. We report
here the presence and incidence of the florfenicol resistance gene,
flo, in avian E. coli.
The characteristics of the avian E. coli isolates are
presented in Table 1. Of the 100 isolates
cultured from litter and from clinical and postmortem material, 11 were
found to be resistant to chloramphenicol by disc diffusion (30 µg).
All 11 chloramphenicol-resistant isolates were multidrug resistant, and
4 of these isolates, avian E. coli isolates 5334, 5790, 5840, and 6468, were also resistant to florfenicol by disc diffusion
(<21-mm-diameter zone of inhibition with a 30-µg disc). S. enterica serovar Typhimurium DT104, for which the MIC of
florfenicol was 64 µg/ml, was used as the positive control
(4). E. coli K-12 containing
cmlA (3) exhibited a florfenicol MIC of 2 µg/ml, as did E. coli DH5
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Detection of Florfenicol Resistance Genes in
Escherichia coli Isolated from Sick Chickens
ABSTRACT
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, the negative control. All of
the flo-containing isolates exhibited florfenicol MICs of at
least 32 µg/ml. The florfenicol MIC for two isolates which did not
contain flo was 8 µg/ml. The breakpoints for florfenicol resistance recently adopted for bovine respiratory pathogens are 2 (sensitive), 4 (intermediate), and 8 (resistant) µg/ml (T. Shyrock, Chairholder, Veterinary Antimicrobial Susceptibility
Testing Subcommittee, Nation Committee for Clinical Laboratory
Standards, personal communication, 1999).
TABLE 1.
Characteristics of chloramphenicol-resistant avian
E. colia isolates
The florfenicol-resistant E. coli isolates came from clinical samples sent from different poultry farms in Georgia and North Carolina. To determine whether they represent dissemination of a clonal strain, random amplification of polymorphic DNA (RAPD) by the method of Maurer et al. was employed (17). RAPD analysis showed four distinct patterns, or RAPD types (data not shown), suggesting that florfenicol resistance is not limited to a particular strain of avian E. coli.
DNA-DNA colony hybridizations with probes specific for cmlA,
flo, and int were done to correlate the presence
of these genes with florfenicol resistance. PCR was used to generate
the DNA probes; Table 2 lists the primers
employed and the expected sizes of the PCR products. The identities of
the PCR products were confirmed by DNA sequencing. The PCR mixture
consisted of 2 mM MgCl2, 0.2 mM deoxyribonucleoside
triphosphates (digoxigenin labeled), 50 pmol of each oligonucleotide
primer, and 0.5 U of Taq polymerase (Boehringer Mannheim,
Indianapolis, Ind.). The program parameters for the hot-air
thermocycler were 30 cycles of (i) 94°C for 1 s, (ii) 40°C for 1 s,
and (iii) 72°C for 15 s. The PCR products were purified by using
WIZARD DNA Clean-Up System kits (Promega) and combined with
hybridization buffer, containing 0.75 M sodium chloride, 1% nonfat dry
milk, 0.1% N-laurylsarcosine, and 0.2% sodium dodecyl
sulfate; they were kept frozen at 
20°C until use. Bacterial cells
were patched onto nylon membranes with toothpicks, and DNA-DNA
hybridizations were performed as described by Sambrook et al.
(20), with hybridizations and washes being done at 68°C. Hybridizing DNA fragments were detected by using an anti-digoxigenin antibody-alkaline phosphatase conjugate with a color substrate solution
of 4-nitroblue tetrazolium chloride and
5-bromo-4-chloro-3-indolylphosphate (XP).
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Results of the DNA colony hybridizations are shown in Table 1. Only one isolate, E. coli 6468, contained cmlA, whereas all four florfenicol-resistant isolates contained flo. The int gene probe revealed that 9 of 11 isolates were positive for the int sequence, indicating that integron-related genes were commonly present in multidrug-resistant clinical isolates. DNA-DNA hybridization was also used to assess whether the flo gene was plasmid associated. The flo gene was used to probe plasmids isolated from the flo-positive avian E. coli isolates (Fig. 1). Plasmid DNA was isolated by the S1 nuclease method of Barton et al. (2) and separated by pulsed-field gel electrophoresis (pulse time, 2 to 40 s; voltage, 6 V/cm; 25 h). The DNA was transferred from the agarose gel to a nylon membrane with a vacuum blotter (Bio-Rad, Hercules, Calif.) according to the manufacturer's recommendations. The procedure for DNA-DNA hybridizations was performed as described above. Three of the four isolates contained flo on high-molecular-weight plasmids of 186 and 204 kb. The florfenicol resistance determinant appears to be present in a variety of large-molecular-weight XbaI DNA fragments in avian E. coli, in contrast to the mapping of the flo resistance gene to a 10-kb XbaI fragment of S. enterica serovar Typhimurium DT104 (Fig. 1). Therefore, its location in avian E. coli may be similar to its placement in large-molecular-weight R plasmids in Pasteurella piscicida (14); however, the differences in sizes of the plasmids and fragments suggest that the gene was independently acquired.
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Two of the four florfenicol-resistant isolates also contained int, the DNA integrase gene that is characteristic of integrons, which are transmissible elements deemed important in the horizontal transfer of antibiotic resistance genes. In Salmonella strain DT104, flo is chromosomally located between two integrons (6). Many of the antibiotic resistance genes found in gram-negative bacteria are located within integrons, which are mobile genetic elements (18). The integrase acts as a site-specific DNA recombinase in the insertion of antibiotic resistance genes into these elements (8, 18). For example, cmlA is present within the integron of Tn1696, which makes up part of the Pseudomonas aeruginosa IncP plasmid R1033 (3). The cmlA drug resistance gene does not appear to be responsible for high-level florfenicol resistance, since we found that only one florfenicol-resistant E. coli isolate possessed the gene and since E. coli containing cmlA was sensitive to florfenicol.
Our study demonstrates the persistence of chloramphenicol resistance in avian E. coli, although this drug has not been used therapeutically in food animals since its use was officially banned in 1988 (13). We also demonstrated that a low percentage (4%) of clinical avian E. coli isolates already display resistance to florfenicol, although the drug is not used therapeutically in chickens. In fact, a feed formulation is not currently available in the United States. Poultry production is rather unique in the United States since the processing company owns the birds and the feed; farmers are contracted to house the animals. The processing company employs veterinarians who are responsible for vaccination and medication of the birds in the face of illness. Therefore, the attending veterinarian prescribes the antibiotics used for treatment of disease, and it is improbable that the birds from which we isolated florfenicol-resistant E. coli had ever been exposed to florfenicol.
Antimicrobials are useful therapeutic agents only if the drug concentrations achieved in the serum and tissue are greater than the MIC of the drug. Florfenicol attains a maximum concentration of 3 µg/ml in the serum of feeder calves (16). The manufacturer of florfenicol reports an MIC of 1 µg/ml or less against 90% of the bacterial isolates from natural infections in cattle (5, 11, 23). Pharmacokinetic studies have shown that the peak plasma florfenicol concentration in ducks and chickens is approximately 3 µg/ml, with similar levels in the liver, kidney, and lung tissues (1, 10, 19). Our study showed that all 11 chloramphenicol-resistant avian E. coli isolates had florfenicol MICs of greater than 3 µg/ml, suggesting that this antimicrobial agent may not be therapeutically successful in some cases.
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ACKNOWLEDGMENTS |
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This study was supported by funding from the Veterinary Medicine Experiment Station.
We thank Karen Jacobsen and Doug Kemp for direction regarding florfenicol usage information and Cynthia Liebert and Barry Harmon for suggestions regarding the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department Medical Microbiology and Parasitology, The University of Georgia, Athens, GA 30602. Phone: (706) 542-5778. Fax: (706) 542-5771. E-mail: leem{at}calc.vet.uga.edu.
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