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

Abstract

ᰔFlorfenicol (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 g/ml) for selection. The antimicrobial susceptibilities of M07-1, the corresponding FFCresistant 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 multidrugresistant (MDR) transconjugant harbored the floR gene.E. ictaluri plasmid pM07-1 also conferred resistance to ceftiofur and concomitant decreased susceptibility to ceftriaxone, implying the presence of the bla CMY-2 beta-lactamase gene. Recent data indicate a rapid increase in the dissemination of cephalosporin resistance linked to self-transmissible MDR plasmids harboring bla 16). Plasmid-borne bla CMY-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 bla CMY-2 was performed to verify the presence of this gene in E. ictaluri

β-Lactam Resistance in Salmonella Strains Isolated from Retail Meats in the United States by the National Antimicrobial Resistance Monitoring System between 2002 and 2006

“…The plasmid backbone of pSN254 showed 99% DNA sequence homology with the IncA/C plasmid backbone commonly present in many members of the Enterobacteriaceae such as Yesinia, Aeromonas, and Vibrio (17,20). This plasmid appears to be nearly universally present in Tio r meat isolates of Salmonella (29) and is also present in MDR E. coli and Klebsiella from retail meats (30) The IncA/C backbone is also similarly detected in Tio r Salmonella isolated from varieties of animal species (16).…”

Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family

“…All antimicrobial resistance determinants are carried within specific, but partially overlapping, mosaic regions that are integrated at few sites within the conserved plasmid backbone. Sequence composition and codon usage deviate in these regions from the conserved plasmid backbone and are indicative of horizontal gene transfer (44). Most resistance determinants are located within resistance gene arrays, composed of resistance genes or gene clusters and mobile genetic elements such as IS elements, transposons or integrons.…”

“…This group of plasmids is regarded as a considerable public health threat due to its ability to spread across taxonomic borders (8,20,29,34,44,45). In this study, we present the complete plasmid sequence of pRA1 and provide new insights into the evolution of MDR IncA/C plasmids that allow for a better understanding of the underlying principles of plasmid propagation.…”

Selection Pressure Required for Long-Term Persistence of bla CMY-2 -Positive IncA/C Plasmids

“…bla CMY-2 is often found on large (ca. 140-to 160-kb) IncA/C plasmids (bla CMY-2 plasmids) from a diverse range of bacterial hosts, including Escherichia, Salmonella, Shigella, Klebsiella, Edwardsiella, and Aeromonas (31,35,50). These bla CMY-2 plasmid-bearing bacteria have been detected in mammals and fish without a documented or consistent pattern of antibiotic selection pressure (2,17,26,31,37,(50)(51)(52).…”

“…140-to 160-kb) IncA/C plasmids (bla CMY-2 plasmids) from a diverse range of bacterial hosts, including Escherichia, Salmonella, Shigella, Klebsiella, Edwardsiella, and Aeromonas (31,35,50). These bla CMY-2 plasmid-bearing bacteria have been detected in mammals and fish without a documented or consistent pattern of antibiotic selection pressure (2,17,26,31,37,(50)(51)(52). There is a high prevalence (up to 100%) of bla CMY-2 plasmids reported in commensal and pathogenic bacteria such as Escherichia coli and Salmonella enterica in cattle herds across the United States (1,15,30,42,51).…”