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Antimicrobial Agents and Chemotherapy, March 2009, p. 1256-1259, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01284-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

CMY-31 and CMY-36 Cephalosporinases Encoded by ColE1-Like Plasmids{triangledown}

A. Zioga,1 J. M. Whichard,2 S. D. Kotsakis,1 L. S. Tzouvelekis,3 E. Tzelepi,1 and V. Miriagou1*

Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece,1 Division of Foodborne, Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,2 Department of Microbiology, School of Medicine, University of Athens, Athens, Greece3

Received 24 September 2008/ Returned for modification 21 November 2008/ Accepted 13 December 2008


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ABSTRACT
 
Two CMY-2 derivatives, CMY-31 (Gln215->Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala77->Cys and Gln193->Glu) from Klebsiella pneumoniae, were characterized. Both cephalosporinases functionally resembled CMY-2. blaCMY alleles occurred as parts of a putative transposon comprising ISEcp1B and a Citrobacter freundii-derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca.


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INTRODUCTION
 
Various plasmid-mediated cephalosporinases, mainly from enterobacteria, have been described. Producers of these enzymes are commonly resistant to penicillins, penicillin-inhibitor combinations, narrow-spectrum cephalosporins, and cefoxitin. Activities of expanded-spectrum cephalosporins (ESCs) are also compromised depending on the amount of cephalosporinase (3, 17). Plasmid-mediated cephalosporinases are classified into five evolutionary groups, the most widespread being the group of Citrobacter freundii-derived enzymes, including CMY-2 and more than 30 variants (17; www.lahey.org/Studies/). The blaCMY-2 alleles are encountered in both hospital- and community-acquired enterobacteria worldwide. Notably, they have emerged also in nontyphoid salmonellae, including Salmonella enterica serotype Newport, currently spreading epidemically in the United States (9, 14, 17). The important role of IncA/C and IncI plasmids in the diffusion of CMYs has been documented (10). A CMY-5-encoding ColE1-like plasmid has also been described (24). We report here on two CMY variants, CMY-31 and CMY-36, encoded by ColE1-like plasmids.


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CMY-producing microorganisms.
 
Serotype Newport AM17274 was submitted to the National Antibiotic Resistance Monitoring System (CDC, Atlanta, GA) in 2003 from Nevada. Klebsiella pneumoniae HP205 was recovered in a hospital in Athens, Greece, in 2005. β-Lactam MICs, determined by agar dilution, indicated cephalosporinase production, i.e., resistance to penicillins, penicillin-inhibitor combinations, cefoxitin, and ESCs and susceptibility to cefepime and imipenem (Table 1). Isolates exhibited resistance also to sulfonamides and streptomycin. K. pneumoniae HP205 was, additionally, resistant to trimethoprim, gentamicin, and tobramycin. Isolates were positive in a blaCMY-2-specific PCR (6) and produced β-lactamases with alkaline isoelectric points (pIs > 8.5 as determined by isoelectric focusing) that, most likely, represented the CMY cephalosporinases. K. pneumoniae HP205 also produced a β-lactamase with a pI of 7.6, consistent with the resident β-lactamase of this species.


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  		TABLE 1. β-Lactam susceptibility of the CMY-producing clinical isolates serotype Newport AM17274 and K. pneumoniae HP205 and E. coli DH5{alpha} clones carrying the wild-type plasmids pA172 and pH205 and the respective CMY-encoding recombinant plasmids


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Characterization of CMY-31 and CMY-36.
 
Plasmids from AM17274 and HP205 were isolated with a Midi kit (Qiagen, Hilden, Germany) and introduced into Escherichia coli DH5{alpha} by transformation, yielding cephalosporin-resistant clones (Table 1) containing blaCMY-2-type genes located in similar plasmids, pA172 from serotype Newport and pH205 from K. pneumoniae. Entire plasmid sequences were determined on both strands by a primer walking approach using an API377 sequencer (Applied Biosystems, Foster City, CA). Sequencing data showed that the blaCMY gene in pA172 differed by a single nucleotide from blaCMY-2 (GenBank accession no. X91840), resulting in a Gln215->Arg substitution. An identical gene (blaCMY-31) has recently been identified in K. pneumoniae in Switzerland (GenBank accession no. EF622224) (1). In pH205 the respective gene differed from blaCMY-2 by 7 nucleotides (nt), and the putative novel cephalosporinase (CMY-36) would include the substitutions Ala77->Cys and Gln193->Glu compared to CMY-2. Each CMY protein comprised 381 amino acids, including a 20-amino-acid putative signal peptide. The calculated pIs (9.02 for CMY-31 and 8.94 for CMY-36) were in accordance with those determined by isoelectric focusing (9.2 and 9.0, respectively). Based on the structure of the Enterobacter cloacae 908R β-lactamase (PDB entry 1Y54) (13), it seems that Arg215 is located in the {Omega} loop that is involved in the interaction with β-lactam substrates (15). Both substitutions in CMY-36 occur at positions distant from the active site cavity (Fig. 1).


Figure 1
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  		FIG. 1. Ribbon diagram based on the AmpC from E. cloacae 908R (PDB entry 1Y54) (13), indicating the likely locations of amino acid residues Arg215 (CMY-31) and Cys77 and Glu193 (CMY-36) in relation to the active-site cavity indicated by the conserved residues Ser64, Tyr150, and Lys315. The image was made using the DeepView/Swiss-Pdb viewer, version 3.7, available at www.expasy.org/spdbv/.

The entire blaCMY-type genes from pA172 and pH205, along with the ISEcp1B-located promoter (8), were amplified by PCR using the primers ecp-F2 (5'-CGGAATTCGTTGCTCTGTGGATAACTTG-3') and ampC2 (5'-CGGAATTCTTGCAGCTTTTCAAGAATGCGC-3') (nt 2084 to 2103 and 3496 to 3517, respectively [GenBank accession no. EU331425 and EU331426, respectively]), which also contained EcoRI restriction sites (underlined). Amplicons (1,434 bp) were restricted with EcoRI and ligated into the high-copy-number vector pBCSK(+) (Stratagene, La Jolla, CA), resulting in plasmids pB-cmy31 and pB-cmy36. Plasmid pB-cmy31 was utilized to construct a similar blaCMY-2-carrying plasmid (pB-cmy2) by site-directed mutagenesis with a QuikChange mutagenesis kit (Stratagene) and the mutagenic primers cmF (5'-TTTCTCCGGGACAACTTGACGCCGA-3') and cmR (5'-TCGGCGTCAAGTTGTCCCGGAGAAA-3') (nt 3066 to 3090 in EU331425; G->A [underlined] in codon 215, resulting in Arg->Gln). Mutations were verified by sequencing at least two independently amplified PCR products.

Plasmids pB-cmy31, pB-cmy36, and pB-cmy2 were introduced into E. coli DH5{alpha}. β-Lactam MICs showed that the three CMY variants conferred comparable levels of resistance to cefotaxime, ceftazidime, ceftriaxone, piperacillin, and piperacillin-tazobactam. MICs of ampicillin, amoxicillin (amoxicilline)-clavulanate, ticarcillin, and cefoxitin, however, exceeded the range of dilutions tested. The strains were susceptible to cefepime and imipenem (Table 1).

Substrate and inhibition profiles of the CMY enzymes were also determined. Cell suspensions of the E. coli DH5{alpha} clones carrying pB-cmy31, pB-cmy36, and pB-cmy2 were sonicated, and extracts were clarified by ultracentrifugation. Cephalosporinase purification was performed by two ion-exchange chromatography steps using Q- and S-Sepharose (16). Purity of the preparations was >90%, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic parameters for penicillin G, ampicillin, cephaloridine, cephalothin (cefalotin), nitrocefin, and cefotaxime hydrolysis were studied by spectrophotometry (16). Interaction of CMY-31 and CMY-36 with β-lactam substrates was typical for enterobacterial AmpCs (i.e., rapid hydrolysis of early-generation cephalosporins and significantly lower rates of hydrolysis of penicillins and oxyimino-cephalosporins). Kinetic parameters of the two CMY enzymes did not differ significantly from those of CMY-2, determined in parallel (Table 2). Inhibitory activities of cloxacillin, aztreonam, and Ro 48-1220 against CMY-31, CMY-36, and CMY-2 were studied by UV spectrophotometry using cephalothin (100 µM) as a reporter substrate. Results were expressed as 50% inhibitory concentrations (IC50s). Inhibition profiles for the three CMY variants were similar. Both cloxacillin and aztreonam were potent inhibitors (IC50s were 0.004 and 0.006 to 0.007 µM, respectively). IC50 values for Ro 48-1220 ranged from 0.3 to 0.35 µM. Taken together with the similar MICs under isogenic conditions, these data indicated that CMY-31 and CMY-36 are functionally similar to CMY-2.


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  		TABLE 2. Kinetic parameters of CMY-type cephalosporinases for various β-lactam substratesa


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Characteristics of CMY-encoding plasmids.
 
pA172 and pH205 each comprised 8,197 bp and differed at 33 and 40 nt positions, respectively, from pTKH11 (GenBank accession no. Y17716), a ColE1-like blaCMY-5-carrying plasmid from K. oxytoca isolated in Sweden in 1991 (24). Sequence annotation for pA172 and pH205 (GenBank accession no. EU331425 and EU331426) complements that proposed for pTKH11 (24) by including recently characterized ISEcp1B (18) as well as the oriT and cer regions.

Each plasmid carried an ISEcp1B-associated C. freundii-derived sequence of 2,088 bp (blaCMY, blc [encoding lipocalin], and sugE [encoding a small multidrug resistance protein]) flanked by 5-bp target site duplications (5'-GATTA-3'; nt 604 to 608 and 4634 to 4638 in EU331425 and EU331426) and inserted into a ColE1-like backbone of 4,167 bp. An oriV (nt 1599 to 1601) was located 40 bp from the regulatory elements RNA II (511 bases) and RNA I (105 bases) (4). ORF7 (471 bp), downstream of RNA I, resembled a remnant (mobB) of the mob gene array of ColE1 (23). ORF6 (594 bp), of unknown function, was located between mobB and the CMY-encoding element. An AT-rich 227-bp sequence (nt 7094 to 7311) exhibiting 81% homology with the transfer origin (oriT) from pIP843 and pJHCMW1 (5, 20) and a 266-bp sequence (nt 7964 to 8182) 95% homologous to the cer (ColE1 resolution) recombination site implicated in plasmid monomerization and stability (22) were also identified (Fig. 2).


Figure 2
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  		FIG. 2. Genetic organization of the ColE1-like plasmids pA172 from Salmonella serotype Newport and pH205 from K. pneumoniae encoding CMY-2-type cephalosporinases. Arrows show directions of transcription of open reading frames and regulatory elements. Locations of the ColE1 sequences oriV, oriT, and cer are indicated. The gene mobB was reported as ORF7 in the CMY-5-encoding plasmid pTKH11 (21). Target site duplications (TSDs) at the boundaries of the CMY-encoding transposon and the respective inverted repeats (IRR and IRL) are also shown.

To test their mobilization capability, plasmids pA172 and pH205 were introduced into E. coli XL1-Blue, which harbors fertility factor F', an IncFIA conjugative plasmid. The resulting transformants were able to transfer the CMY-encoding plasmids to E. coli DH5a during conjugation at a relatively low frequency (10–7 blaCMY-positive clones per donor cell).


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Conclusions.
 
Most plasmid-mediated blaCMY genes have likely originated from blaCMY-2 through point mutations, leading to the present array of CMY variants (2). CMY-31 and CMY-36 exhibit activities similar to that of CMY-2. Therefore, the respective amino acid changes, including Gln215->Arg in the {Omega} loop, are probably not associated with selective pressure exerted by drugs such as ESCs. The common-origin hypothesis of the plasmid-borne blaCMY alleles is also in line with the similarity of their genetic environments. The elements from the ColE1-like plasmids, as well as a similar CMY-2-encoding transposon from an IncI plasmid (21), occur as shorter versions of ISEcp1B-associated sequences from a variety of plasmids (8, 10, 11). It can be hypothesized that these transposon-like sequences are derived from a common element through alterations in the right-hand end that may arise during ISEcp1B-mediated transposition (18, 19). Nevertheless, an alternative evolutionary route, such as multiple transposition events in a blaCMY-containing sequence from a single C. freundii strain, cannot be ruled out.

A possible role for a ColE1-like plasmid in the global spread of CMYs is also indicated. This plasmid species was probably formed through transposition of a C. freundii-originating sequence into a ColE1 replicon and diverged by point mutations. It was first identified in clinical Enterobacteriaceae in the early 1990s in Sweden (24) and Greece (7; our unpublished data) and, as indicated here, persists in the latter country. Notably, the CMY-encoding ColE1-like plasmid has also been acquired by an important food-borne pathogen, Salmonella serotype Newport. ColE1 can be mobilized by plasmids with apparently different conjugation systems such as InFI, IncI1, and IncP. Mobilization efficiency depends on the proteins encoded by the mob genes of ColE1 (23). Nevertheless, there have been examples of mobilization of ColE1 derivatives, in which, as in the ones described here, the full array of mob genes are deleted. The presence of an oriT sequence may play a role in this process (12). Whichever the mechanism, the in vitro mobilization shown here and the carriage by different bacterial species underscore the spreading potential of these CMY-encoding ColE1-like plasmids.


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ACKNOWLEDGMENTS
 
This work was supported by the Hellenic Pasteur Institute and Kapodistrias grant 70/4/9103 from the University of Athens. The National Antibiotic Resistance Monitoring System is funded through an interagency agreement between the U.S. Food and Drug Administration Center for Veterinary Medicine and the Centers for Disease Control and Prevention.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Bacteriology, Hellenic Pasteur Institute, Vas. Sofias 127, Athens 11521, Greece. Phone: 30-210-6478810. Fax: 30-210-6426323. E-mail: miriagou{at}pasteur.gr Back

{triangledown} Published ahead of print on 22 December 2008. Back


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Antimicrobial Agents and Chemotherapy, March 2009, p. 1256-1259, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01284-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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  • Carattoli, A. (2009). Resistance Plasmid Families in Enterobacteriaceae. Antimicrob. Agents Chemother. 53: 2227-2238 [Full Text]  

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