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Antimicrobial Agents and Chemotherapy, February 2009, p. 845-846, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01312-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

IncA/C Plasmid-Mediated Florfenicol Resistance in the Catfish Pathogen Edwardsiella ictaluri{triangledown}


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LETTER
 
Florfenicol (FFC) has recently been approved by the Food and Drug Administration for the treatment of several bacterial diseases of cultured fish species in the United States, including enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri. The FFC-resistant E. ictaluri strain (M07-1) described herein was isolated from a moribund catfish obtained from the Thad Cochran National Warmwater Aquaculture Research Center (Stoneville, MS) in May of 2007 and was confirmed to be E. ictaluri by 16S rRNA gene sequencing (6). Fish showing signs of ESC were examined for FFC-resistant E. ictaluri because losses due to ESC persisted in this population despite FFC treatment. To characterize the resistance properties of this strain, conjugative transfer experiments were performed as described previously (14) using FFC (30 µg/ml) for selection. The antimicrobial susceptibilities of M07-1, the corresponding FFC-resistant transconjugant, and the isogenic parent strain (Table 1) were quantified using standard microdilution assays (3, 4, 11), demonstrating that the resistance phenotype observed in strain M07-1 was self-transmissible, conferring resistance to FFC, chloramphenicol, tetracycline, streptomycin, ampicillin, amoxicillin-clavulanic acid, ceftiofur, and cefoxitin, as well as decreased susceptibility to trimethoprim-sulfamethoxazole and ceftriaxone. Resistance transfer correlated to a 150-kb plasmid (referred to hereinafter as pM07-1), suggesting the presence of a multidrug resistance plasmid in this isolate (data not shown). PCR analysis (15) followed by sequencing confirmed that E. ictaluri M07-1 and its multidrug-resistant (MDR) transconjugant harbored the floR gene.


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  		TABLE 1. Antimicrobial susceptibilities of E. ictaluri M07-1 and its E. coli transconjugant

E. ictaluri plasmid pM07-1 also conferred resistance to ceftiofur and concomitant decreased susceptibility to ceftriaxone, implying the presence of the blaCMY-2 beta-lactamase gene. Recent data indicate a rapid increase in the dissemination of cephalosporin resistance linked to self-transmissible MDR plasmids harboring blaCMY-2 (13, 16). Plasmid-borne blaCMY-2 genes in bacteria isolated from food animals, retail meats, and humans in the United States have been identified previously (1, 2, 5, 8, 16) but have not been detected before in bacteria associated with U.S. aquaculture and were not expected, as there are currently no cephalosporin antimicrobials approved for use in U.S. aquaculture. Therefore, PCR analysis using previously described primers specific to blaCMY-2 was performed to verify the presence of this gene in E. ictaluri M07-1 and its correlation with plasmid pM07-1 transfer. The blaCMY-2 amplicon was detected in M07-1 and in the corresponding FFC-selected Escherichia coli transconjugant and was 100% identical to previously described plasmid-carried blaCMY-2 genes in Salmonella enterica, E. coli, and Klebsiella pneumoniae.

There have been several reports of IncA/C plasmids from various Salmonella and E. coli strains that harbor the blaCMY-2 gene and more recent reports of IncA/C plasmids harboring both blaCMY-2 and floR in human clinical isolates of Salmonella serovar Newport (7, 14) and in an isolate of the fish pathogen Aeromonas salmonicida subsp. salmonicida from Atlantic salmon in Canada (10). IncA/C MDR plasmids have also been identified in a human clinical isolate of Yersinia pestis from Madagascar and in several clinical isolates of Vibrio cholerae O139 from China (12, 14). Plasmid pM07-1 was evaluated for the presence of the IncA/C backbone by using 13 backbone-specific PCR assays as described previously (14). Both the M07-1 parent strain and the E. coli transconjugant harboring pM07-1 were positive for all 13 IncA/C plasmid markers examined, thus providing definitive evidence that plasmid pM07-1 is a member of the IncA/C family.

This report documents the first case of FFC resistance in the catfish pathogen E. ictaluri. Interpretive standards for FFC sensitivity testing of E. ictaluri have not yet been established; however, the MIC for E. ictaluri M07-1 (128 µg/ml) was approximately 500 times higher than the average MIC determined for susceptible strains of E. ictaluri (9), suggesting that strains carrying plasmid pM07-1 would be less likely to respond clinically to FFC treatment. It is also important to emphasize that plasmid pM07-1 confers decreased susceptibility to all three antimicrobial drugs that are currently approved for use in aquaculture in the United States (FFC, ormetoprim-sulfadimethoxine, and oxytetracycline) and that, therefore, should further dissemination of the pM07-1 plasmid occur, it could have a profound effect on the performance of therapeutic antimicrobial drugs in U.S. aquaculture.


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Nucleotide sequence accession numbers.
 
The sequences of floR and blaCMY-2 determined in this study have been deposited in GenBank under accession numbers FJ438528 and FJ438529.


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ACKNOWLEDGMENTS
 
This work was supported by federal funds from the U.S. Department of Agriculture through ARS CRIS project 1930-31000-002.


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FOOTNOTES
 
{triangledown} Published ahead of print on 24 November 2008. Back


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Timothy J. Welch*
Jason Evenhuis
National Center for Cool and Cold Water Aquaculture
USDA Agricultural Research Service
11861 Leetown Road
Kearneysville, West Virginia 25430

David G. White
Patrick F. McDermott
Heather Harbottle
Ron A. Miller
Center for Veterinary Medicine
Food and Drug Administration
Laurel, Maryland 20708

Matt Griffin
David Wise
Thad Cochran National Warmwater Aquaculture Center
Mississippi State University
Stoneville, Mississippi 38776

* Phone: (304) 724-8340, Fax: (304) 725-0351, Email: E-mail: tim.welch{at}ars.usda.gov

Antimicrobial Agents and Chemotherapy, February 2009, p. 845-846, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01312-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Fricke, W. F., Welch, T. J., McDermott, P. F., Mammel, M. K., LeClerc, J. E., White, D. G., Cebula, T. A., Ravel, J. (2009). Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family. J. Bacteriol. 191: 4750-4757 [Abstract] [Full Text]  

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