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Antimicrobial Agents and Chemotherapy, October 2004, p . 4047-4049, Vol . 48, No . 10
Relationship between Phenotypic and Genotypic Florfenicol Resistance in Escherichia coli
Randall S . Singer,1,2* Sheila K . Patterson,1 Anne E . Meier,1 Jessica K . Gibson,1 Hannah L . Lee,1 and Carol W . Maddox1,3
Department of Veterinary Pathobiology,1
Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, Illinois,3
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St . Paul, Minnesota2
Received 1 December 2003/
Returned for modification 3 March 2004/
Accepted 31 May 2004
This study evaluated the relationship between florfenicol resistance and flo genotypes in 1,987 Escherichia coli isolates from cattle . The flo gene was detected in 164 isolates, all of which expressed resistance to florfenicol at MICs of
 256 µg/ml . The florfenicol MICs for all isolates that lacked flo were
 16 µg/ml .
Florfenicol is a fluorinated analog of chloramphenicol approved by the Food and Drug Administration in 1996 for the treatment of bovine respiratory disease (BRD) pathogens . The main florfenicol targets in respiratory diseases of cattle are Pasteurella multocida, Mannheimia haemolytica, and Haemophilus somnus . Florfenicol is occasionally used extra-label in the treatment of calf diarrhea (10) and can be effective against Escherichia coli strains isolated from diarrheic calves (9) . Florfenicol binds to the 50S subunit of the bacterial ribosome and disrupts protein synthesis . E . coli strains have been recovered with a gene known as flo that expresses resistance to florfenicol and chloramphenicol and is typically located on large transferable plasmids (7) . Neither the enzyme chloramphenicol acetyltransferase nor the nonenzymatic chloramphenicol resistance gene cmlA confers resistance to florfenicol (10) .
Past studies documented florfenicol resistance in animal isolates of E . coli . White et al . (10) found that for 44 of 48 neonatal calf diarrhea E . coli isolates, there was decreased susceptibility to florfenicol, flo was present, and the florfenicol MIC range was 16 to
256 µg/ml . For 4 of the 48 E . coli isolates, cmlA was present, flo was not present, and the florfenicol MIC range was
8 to 64 µg/ml . Keyes et al . (6) analyzed chloramphenicol-resistant E . coli from sick chickens and found that some isolates possessed the flo gene . The florfenicol MICs for isolates harboring flo were
32 µg/ml, whereas the florfenicol MICs for isolates without flo were
8 µg/ml . Bischoff et al . (1) analyzed 48 chloramphenicol-resistant E . coli isolates from cases of neonatal swine diarrhea . The florfenicol MIC for one isolate, which possessed flo, was 256 µg/ml . The florfenicol MICs for the remaining 47 isolates ranged from 8 to
16 µg/ml .
Currently, there are no National Committee for Clinical Laboratory Standards (NCCLS) MIC breakpoints approved to indicate florfenicol resistance in E . coli, and many studies (1, 10) use MIC breakpoints for the BRD pathogens . When these guidelines are used, an MIC of
8 µg/ml indicates resistance, an MIC of 4 µg/ml indicates intermediate susceptibility, and an MIC of
2 µg/ml indicates susceptibility . Previous studies demonstrated that the presence of flo is associated with very high florfenicol MICs (1, 6, 10) . Therefore, we hypothesized that MIC breakpoints for BRD pathogens would not be useful in correlating the presence of flo in E . coli isolates with the observed florfenicol MIC . The objective of this study was to evaluate the relationship between florfenicol resistance phenotypes and flo genotypes in commensal bovine isolates of E . coli .
During an intensive, longitudinal study of antimicrobial resistance in dairies, 1,987 E . coli isolates were cultured from 195 bovine fecal samples . Cattle of different ages in each of four dairies were sampled every 3 months for 18 months . The antimicrobial MICs of florfenicol and chloramphenicol were determined for each isolate by use of broth microdilution in accordance with NCCLS guidelines (8) . Antibiotic concentrations ranged from 0.5 to 512 µg/ml . The MIC was defined as the lowest concentration of antibiotic completely inhibiting visible growth . E . coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains . The florfenicol MIC distribution showed a clear bimodal pattern (Fig . 1), with the MICs for all isolates being either
16 or
256 µg/ml . The distribution for chloramphenicol MICs had a similar pattern, although isolates were identified along the entire continuum of MICs .
 | FIG . 1 . Histogram depicting the distribution of florfenicol (FLO) and chloramphenicol (CLM) MICs for the 1,987 E . coli isolates tested.
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We designed and optimized a multiplex PCR assay (Table 1) for the detection of the flo and cmlA genes . This assay included E . coli 16S rRNA primers as the DNA template quality control . Both the flo and cmlA genes were detected in the E . coli isolates in this study (Table 2) . We did not observe any isolates that possessed the cmlA gene but lacked the flo gene . All of the isolates for which the florfenicol MICs were
256 µg/ml possessed the flo gene; no isolates for which the florfenicol MIC was
16 µg/ml possessed the flo gene (Fig . 1) . The isolates that possessed the flo gene came from 60 different animals, and all four farms were represented . Some animals had flo-positive E . coli isolates on more than one sampling .
| TABLE 1 . Primers and conditions used in PCR
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| TABLE 2 . Prevalence of flo and cmlA genes in the 1,987 E . coli isolates tested
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All 99 flo-positive isolates from one farm were tested to determine if the flo gene resided on conjugative plasmids . Conjugation was carried out by plate mating, with a nalidixic acid-resistant E . coli strain (DH5 ) as the recipient . Thirty-seven of the isolates (37.3%) were able, by conjugation, to transfer resistance to florfenicol . The remaining isolates may have the flo gene located on a chromosome or on nonconjugative plasmids (10) .
All E . coli isolates were genotyped by repetitive-element PCR fingerprinting (Table 1) using the BOXA1R primer and a touchdown program similar to that described by Johnson and O'Bryan (4) . For quality control, a standard lab strain was amplified in each set of reactions to demonstrate repeatability and consistency across all experiments . Dendrograms were created by use of BioNumerics 3.0 software with the Dice coefficient (3) and the neighbor-joining algorithm . Sixty-six different E . coli repetitive-element PCR fingerprint patterns were found among the flo-positive isolates . Some flo-positive fingerprint patterns persisted over several samplings . No fingerprint patterns were shared between E . coli isolates that could transfer florfenicol resistance by conjugation and those that could not . This study corroborated the findings of other studies (1, 6, 10) in which a diverse array of E . coli strains possessed a flo gene carried on conjugative plasmids in many of the isolates .
The florfenicol MICs for many chloramphenicol-resistant, flo-negative E . coli isolates would be considered as indicating resistance if the breakpoints for BRD pathogens were used . According to this classification, 476 (24.1%) flo-negative E . coli isolates from this study would be considered florfenicol resistant . Other studies had similar findings in which flo-negative E . coli isolates would be considered florfenicol resistant (1, 6, 10) . Some investigators have suggested that perhaps another mechanism confers an intermediate florfenicol MIC in the range of 8 to 32 µg/ml (1, 10) . Research studies or surveillance systems might overestimate florfenicol resistance if they were to use the BRD pathogen MIC breakpoints for E . coli isolates . In the case of florfenicol resistance, the resistance phenotype is often used as a surrogate for the underlying genetic mechanism conferring the resistance, which in this case is the presence of the flo gene . If the goal of the resistance classification system is to have some relationship to the likely genetic mechanisms for resistance, then florfenicol resistance in E . coli might be defined by an MIC of
32 µg/ml . In our work and in other studies, an MIC of
32 µg/ml rarely would have been confused with a chloramphenicol resistance mechanism . Clinical relevance of an MIC breakpoint of 32 µg/ml would need to be examined .
The research in this project has complied with all relevant federal animal use guidelines and institutional policies .
This project was supported by USDA National Research Initiative competitive grant 00-35212-9398 (R . S . Singer) .
We thank Elizabeth Lyle and Heather Estilo for technical assistance and Richard Wallace for his involvement with the study .
* Corresponding author . Mailing address: Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 300A VSB, 1971 Commonwealth Ave., St . Paul, MN 55108 . Phone: (612) 625-6271 . Fax: (612) 625-5203 . E-mail: singe024{at}umn.edu .
- Bischoff, K . M., D . G . White, P . F . McDermott, S . Zhao, S . Gaines, J . J . Maurer, and D . J . Nisbet. 2002 . Characterization of chloramphenicol resistance in beta-hemolytic Escherichia coli associated with diarrhea in neonatal swine . J . Clin . Microbiol . 40:389-394.
- Bolton, L . F., L . C . Kelley, M . D . Lee, P . J . Fedorka-Cray, and J . J . Maurer. 1999 . Detection of multidrug-resistant Salmonella enterica serotype typhimurium DT104 based on a gene which confers cross-resistance to florfenicol and chloramphenicol . J . Clin . Microbiol . 37:1348-1351.
- Dice, L . R. 1945 . Measures of the amount of ecological association between species . Ecology 26:297-302.
- Johnson, J . R., and T . T . O'Bryan. 2000 . Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli . Clin . Diagn . Lab . Immunol . 7:265-273.
- Kariyama, R., R . Mitsuhata, J . W . Chow, D . B . Clewell, and H . Kumon. 2000 . Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci . J . Clin . Microbiol . 38:3092-3095.
- Keyes, K., C . Hudson, J . J . Maurer, S . Thayer, D . G . White, and M . D . Lee. 2000 . Detection of florfenicol resistance genes in Escherichia coli isolated from sick chickens . Antimicrob . Agents Chemother . 44:421-424.
- Meunier, D., S . Baucheron, E . Chaslus-Dancla, J . L . Martel, and A . Cloeckaert. 2003 . Florfenicol resistance in Salmonella enterica serovar Newport mediated by a plasmid related to R55 from Klebsiella pneumoniae . J . Antimicrob . Chemother . 51:1007-1009.
- National Committee for Clinical Laboratory Standards. 2003 . Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals . Approved standard M31-A, 2nd ed . National Committee for Clinical Laboratory Standards, Wayne, Pa.
- Orden, J . A., J . A . Ruiz-Santa-Quiteria, S . Garcia, D . Cid, and R . De La Fuente. 2000 . In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic dairy calves to 15 antimicrobial agents . J . Vet . Med . B 47:329-335.
- White, D . G., C . Hudson, J . J . Maurer, S . Ayers, S . Zhao, M . D . Lee, L . Bolton, T . Foley, and J . Sherwood. 2000 . Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea . J . Clin . Microbiol . 38:4593-4598.
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