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Antimicrobial Agents and Chemotherapy, April 2006, p. 1590-1593, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1590-1593.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Variability in the Region Downstream of the blaCMY-2 ß-Lactamase Gene in Escherichia coli and Salmonella enterica Plasmids

Min-Su Kang, Thomas E. Besser, and Douglas R. Call*

Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington

Received 15 August 2005/ Returned for modification 2 October 2005/ Accepted 13 January 2006


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ABSTRACT
 
We used sequencing and PCR mapping to investigate the regions flanking the blaCMY-2 ß-lactamase gene carried by plasmids from Escherichia coli and Salmonella enterica. Results revealed genetic heterogeneity in the region downstream from blaCMY-2 and identified a putative transposable element bearing blaCMY-2.


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TEXT
 
The blaCMY-2 gene encodes an AmpC-like ß-lactamase and has been detected on plasmids in a variety of multidrug-resistant Escherichia coli and nontyphoidal Salmonella isolates from humans and animals in the United States (3, 19, 21, 23). The CMY-2 ß-lactamase mediates resistance to expanded-spectrum cephalosporins (e.g., ceftriaxone) that are commonly used to treat salmonella infections in children as these drugs are safe and efficacious (8, 9). Recent data indicate that clonal dissemination of bacteria carrying blaCMY-2-bearing plasmids and the horizontal transfer of a blaCMY-2-bearing plasmid among different bacteria have both contributed to the dissemination of blaCMY-2 (1, 22). In addition, plasmid analysis and molecular hybridization techniques suggest that blaCMY-2 can be transferred among different bacterial strains, including Salmonella and E. coli strains, from food animals and humans by intra- and interspecific plasmid transfer as well as by independent acquisition of blaCMY-2 by different plasmid backbones (7, 16, 20).

Giles et al. provided a partial description of the regions flanking the blaCMY-2 gene for three plasmid types (B, C, and D) found in Salmonella isolates (10). In another study, the blaCMY-2-bearing plasmids from clinical human and animal isolates of E. coli and Salmonella enterica were characterized using mixed-plasmid microarrays. In that study, 21 probe sequences were present in at least 10 of 13 blaCMY-2-positive plasmids comprising two different genotypic clusters (6). Both clusters included plasmids from both E. coli and S. enterica, which was consistent with the intergeneric movement of plasmids, and genetic diversity within clusters was also consistent with the horizontal movement of a putative blaCMY-2-bearing element between distinct plasmids.

In this study, we investigated the genetic context of the blaCMY-2 gene in plasmids from different sources. Twenty-one multidrug-resistant E. coli and S. enterica isolates resistant to expanded-spectrum cephalosporins and cephamycins were used in this study. This collection included 10 isolates that were identified as carrying blaCMY-2-bearing plasmids in a previous study (6) and 11 isolates that were selected from human and animal isolates maintained by the Field Disease Investigation Unit at Washington State University (Pullman, WA) (Table 1). Only one isolate per herd or case was included to maximize the independence between isolates. Isolates were screened for the presence of the blaCMY-2 gene using PCR as previously described (23). Antimicrobial resistance profiles were determined by disk diffusion according to the Clinical and Laboratory Standards Institute guidelines (14). The location of the blaCMY-2 gene was determined by transforming plasmid minipreps into E. coli DH10B cells (GeneHogs; Invitrogen), screening for AmpC-like ß-lactamase-producing transformants on LB media with 50 µg/ml cefoxitin, and conducting plasmid profiling as previously described (12). The presence of the blaCMY-2 gene on the plasmids was also confirmed using PCR as described above. An E. coli DH10B transformant (GH/ps6668) carrying a blaCMY-2-bearing plasmid (150 kb) from an S. enterica serotype Newport isolate (6668) was used for initial characterization of the putative blaCMY-2-bearing element. Plasmid DNA from the other transformants was used for PCR mapping of their blaCMY-2-containing regions. Overlapping PCR assays were designed to span potential gaps between probe fragments that were highly conserved in blaCMY-2-bearing plasmids from E. coli and S. enterica in the previous study (6). PCR products were cloned using a TA cloning kit (Invitrogen). Two independent clones derived from each PCR were chosen and sequenced on both strands (Amplicon Express, Pullman, WA). Open reading frames (ORFs) were identified using Vector NTI software, and their putative functions were predicted using the BLASTX program from the National Center for Biotechnology Information (2). Regions upstream and downstream of blaCMY-2 elements were detected as described by Giles et al. (10). Large DNA fragments (>3 kb) were amplified using long-distance PCR with Platinum Taq DNA polymerase high fidelity (Invitrogen). The most relevant primer sequences are listed in Table 2.


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  		TABLE 1. Characteristics of E. coli and S. enterica isolates tested for the presence of putative transposable elements bearing blaCMY-2


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  		TABLE 2. PCR primers used to determine the context of the blaCMY-2 sequence

Plasmid transformation showed that blaCMY-2 was located on large plasmids ranging in size from approximately 140 to 160 kb (Table 1). The combination of conventional and long-distance PCRs with the plasmid from isolate 6668 revealed a 13-kb blaCMY-2-bearing region flanked at its ends by two ISEcp1-like insertion sequences (ISs) with opposite orientations (Fig. 1). This structure, named type I element, is flanked by a tandem repeat of a partial region downstream of the left IS, which includes a second copy of the blaCMY-2 gene (Fig. 1). The left IS, located just upstream of the blaCMY-2 gene, has 99.8% sequence identity with ISEcp1B (15), while the oppositely oriented ISEcp1 is truncated at the 3' end. The blaCMY-2 gene flanked by the left IS is followed by three genes, including blc (encoding an outer membrane lipoprotein, lipocalin), sugE (encoding a small multidrug resistance protein), and a truncated form of ecnR (encoding a transcriptional regulatory protein, entericidin R) (5). This region shares an overall 99.4% sequence identity with the plasmid pNF4656 (type C), reported by Giles et al. (10), and 99.9 to 100% sequence identity when the comparison is limited to coding regions. The region downstream includes four additional ORFs of unknown function, whose products showed similarity to hypothetical proteins encoded by Pseudomonas resinovorans plasmid pCAR1 (18), dsbC (encoding a disulfide bond isomerase), and a truncated form of traC (encoding a sex pilus assembly and synthesis protein) (4). The traC gene is extensively truncated at the 3' end and merged with the truncated ISEcp1. This finding suggests that an IS insertion adjacent to traC was accompanied or followed by a deletion involving their 3'-end regions. Subsequent PCR mapping with the other plasmids confirmed the presence of the type I element in nine additional isolates (Table 1), including E. coli and S. enterica isolates. The conserved blaCMY-2-bearing type I element, which is flanked by inversely oriented ISs, may be a composite transposon that contributes to the horizontal transfer of blaCMY-2 (11, 17).


Figure 1
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  		FIG. 1. Maps of conserved genetic structures bearing blaCMY-2 identified in expanded-spectrum, cephalosporin-resistant E. coli and S. enterica isolates. The orientation of each gene or IS is indicated by arrows. ORFs that partially overlap their neighbors are shown on the upper row. ORFs 1 to 6 represent predicted genes of unknown function. Lines below the maps indicate regions amplified by the primers listed in Table 2. IRL, left inverted repeat; IRR; right inverted repeat.

Additional PCR mapping revealed a truncated form (type II) of the type I element in an E. coli isolate, an S. enterica serotype Newport isolate, and four S. enterica serotype Typhimurium isolates (Table 1). This type II structure does not have the right IS in its 3'-end region, unlike the type I element (Fig. 1).

Finally, an 8-kb structure (type III) was identified in five S. enterica serotype Dublin isolates (Table 1). The right end region of the type III structure contains an IS26 (13) with an opposite orientation from the ISEcp1 and is 99.9% identical with that of the previously reported blaCMY-2-bearing pNF4656 (10). This structure also contains two additional ORFs (5 and 6) between IS26 and the truncated ecnR that were not found in pNF4656 (Fig. 1) and whose products are similar to hypothetical proteins encoded by P. resinovorans plasmid pCAR1 (18).

A conserved region approximately 9 kb in size within type I and type II structures was amplified by PCR using primers CMY-2-L and TraC-R (Table 2) and further characterized using restriction fragment length polymorphism analysis with three different restriction endonucleases (BsmFI, BsaI, and BanII; New England Biolabs). Restriction enzyme digestions of the 9-kb PCR products were conducted with 16 E. coli and S. enterica isolates that harbor the type I or type II genetic structure. All amplicons showed identical restriction fragment length polymorphism patterns, with five to seven bands in each digestion, indicating that the sequence of the large region was highly conserved among different genomic backgrounds.

This study demonstrates the presence of three different genetic contexts of the blaCMY-2 gene that are shared among CMY-2 ß-lactamase-encoding plasmids of E. coli and S. enterica and that may be implicated in the dissemination of this important resistance determinant. Although the full extent and mobility of these three structure types and associated regions remain to be determined, these easily distinguishable genetic structures may serve as useful markers to study the dissemination of the blaCMY-2 gene within and between bacterial and animal host populations.

Nucleotide sequence accession number. The GenBank accession number for the type I blaCMY-2 element is DQ164214. The other sequences except for the right end region (1.6 kb) of the type III structure were predicted based on the regions identified by PCR, as shown in Fig. 1. The differential sequence of the type III structure has been deposited under accession number DQ249311.


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ACKNOWLEDGMENTS
 
We thank J. Daniels, A. Khachatryan, S. LaFrentz, and Y. Zhang for technical advice and help and M. Davis for helpful discussion and isolate information. We also thank K. Brayton for helpful comments and critical review of the manuscript.

This study was supported by the Agricultural Animal Health Program, College of Veterinary Medicine, Pullman, WA, and funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. N01-AI-30055, and by USDA-NRICGP 0102147.


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FOOTNOTES
 
* Corresponding author. Mailing address: Dept. Vet. Micro. & Path., Washington State University, 402 Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: (509) 335-8529. E-mail: drcall{at}wsu.edu. Back


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Antimicrobial Agents and Chemotherapy, April 2006, p. 1590-1593, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1590-1593.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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