Characterization of blaCMY-11, an AmpC-type plasmid-mediated ß-lactamase gene in a Korean clinical isolate of Escherichia coli

Sang Hee Leea,*, Jae Young Kimb, Gyu Sang Leea, Seok Ho Cheona, Young Jun Ana, Seok Hoon Jeongc and Kye Joon Leeb

a Department of Genetic Engineering, Youngdong University, Chungbuk 370-701; b Department of Microbiology, College of Natural Sciences, Research Centre for Molecular Microbiology, Seoul National University, Seoul; c Department of Clinical Pathology, Kosin University College of Medicine, Pusan, and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, South Korea


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We report the description of a new plasmid-encoded AmpC-type ß-lactamase gene (blaCMY-11) from Escherichia coli K983802.1 that was isolated from a patient in South Korea suffering from a urinary tract infection. Antibiotic susceptibility testing, plasmid analysis, pI determination, transconjugation and Southern blot analysis were carried out to investigate the resistance mechanism to cefoxitin. PCR, sequencing and sequence analysis were used to identify and analyse the ß-lactamase gene (blaCMY-11) responsible for the cefoxitin resistance. CMY-11 and blaCMY-11 are compared with other class C ß-lactamases and their genes to determine phylogenetic relationships. The cefoxitin-resistance phenotype of E. coli K983802.1 reflects the presence of a large plasmid [pYMG-2 (130 kb)]. A ß-lactamase with a pI value of 8.0 from a transconjugant of E. coli K983802.1 was identified by isoelectric focusing. A 1478 bp DNA fragment from pYMG-2 containing blaCMY-11 was sequenced and an open reading frame coding for a 382 amino acid peptide (CMY-11) was found. Phylogenetic analysis clearly shows that blaCMY-11 belongs to the group of ampC-related bla genes. It is likely that blaCMY-11 evolved from blaCMY-1 via blaCMY-10.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The single most prevalent mechanism responsible for resistance to ß-lactam antibiotics among clinical isolates of the Enterobacteriaceae is the production of ß-lactamases.1 These have been classified into four classes (A–D) according to amino acid sequence identity.2 Plasmid-mediated class A ß-lactamases derived from the commonly found TEM and SHV enzymes have been extensively reported.1 They confer resistance to extended-spectrum cephalosporins and aztreonam, but not to cephamycins, and the enzymes are generally susceptible to inhibition by clavulanic acid. Chromosomally encoded and plasmid-encoded AmpC ß-lactamases, mostly conferring resistance to cephamycins (cefoxitin and cefotetan), penicillins, cephalosporins and ß-lactam–ß-lactamase inhibitor combinations are in class C. Chromosomally encoded AmpC ß-lactamases are produced by several potential pathogens, such as Enterobacter spp., Serratia spp., Citrobacter spp., Morganella spp., Pseudomonas aeruginosa, Acinetobacter baumanii, Hafnia alvei, Ochrobactrum anthropi and Escherichia coli (see Lahey Clinic website: http://www.lahey.org/studies/webt.htm). These enzymes are often expressed at low levels and therefore may not contribute significantly to clinical ß-lactam resistance.3 Plasmid-encoded AmpC ß-lactamases have recently been reported in Klebsiella pneumoniae (CMY-1, CMY-2, CMY-8, MOX-1, MOX-2, FOX-1, FOX-5, LAT-1, LAT-2, LAT-2b, ACT-1, MIR-1 and ACC-1), Klebsiella oxytoca (CMY-5 and FOX-3), E. coli (CMY-4, CMY-6, CMY-7, CMY-9, FOX-2, FOX-4, BIL-1, LAT-3 and LAT-4), Salmonella enteritidis (DHA-1), Proteus mirabilis (CMY-3), Salmonella senftenberg (CMY-2b) and Enterobacter aerogenes (CMY-10) (see The Hall Laboratory website: http://www.rochester.edu/College/BIO/labs/HallLab/AmpC_Phylo.html). These enzymes, except ACC-1, confer resistance to cephamycins, 7-oxyimino- ß-lactams, such as cefotaxime and ceftazidime, and the monobactam aztreonam and, accordingly, are extended-spectrum ß-lactamases. Plasmid-encoded AmpC ß-lactamases are often expressed in large amounts and can pose therapeutic problems.3 They can be encoded by transposons or integrons. In this study we report the characterization of a new plasmid-mediated AmpC ß-lactamase gene from a clinical isolate of E. coli.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains

E. coli K983802.1 was isolated in the Kosin Medical Center (Pusan, South Korea) on 11 June 1998 from a patient suffering from a urinary tract infection. E. coli J53 AziR is resistant to sodium azide. E. coli ATCC25922 was used as the MIC reference strain.

MIC determination

Antibiotic susceptibility was determined on Mueller– Hinton agar (Difco Laboratories, Detroit, MI, USA) containing two-fold serial dilutions of antibiotic and inocula of c. 104 cfu/spot. Plates were incubated at 37°C for 18 h. Clavulanic acid was at 2 mg/L. Antibiotics were from the following suppliers: cefalothin (Sigma-Aldrich, St Louis, MO, USA); cefoxitin (Merck Sharp and Dohme-Chibaret, West Point, PA, USA); co-amoxiclav (Ilsung Pharmaceuticals, Ansan, South Korea); cefamandole (Shin Poong Pharmaceutical, Ansan, South Korea); cefotaxime (Handok Pharmaceuticals, Seoul, South Korea); ceftazidime (Hanmi Pharmaceuticals, Hwasung, South Korea); aztreonam (BMS Pharmaceutical Korea, Seoul, South Korea); and cefotetan (Jeil Pharmaceutical, Youngin, South Korea).

Plasmid isolation and analysis

Plasmid DNA was isolated from E. coli K983802.1 as described previously4 and analysed on 1% (w/v) agarose gels using a Field Inversion Gel electrophoresis (FIGE) Mapper system (Bio-Rad, Hercules, CA, USA). Plasmid DNA was recovered using a Gel Extraction Kit (Genomid, Research Triangle Park, NC, USA).

Plasmid transfer

Equal volumes (4 mL) of cultures of E. coli K983802.1 and E. coli J53 AziR (each at 109 cfu/mL) grown in tryptic soy broth (Difco, Detroit, MI, USA) were mixed. Mixtures were incubated at 37°C for 18 h. Transconjugants were selected on Mueller–Hinton agar containing sodium azide (150 mg/L) and cefoxitin (20 mg/L).

Isoelectric focusing of ß-lactamase

Cell extracts of E. coli K983802.1 and E. coli J53AziR(pYMG-2) transconjugants were prepared by osmotic shock, as described in the pET system manual (Novagen, Madison, WI, USA). Isoelectric focusing (IEF) was carried out in Ready Gel Precast IEF polyacrylamide gel containing ampholine (pH range 3–10) in a Mini-Protein 3 cell according to the manufacturer' instructions (Bio-Rad, Hercules, CA, USA). Gels were developed with 0.5 mM nitrocefin (Oxoid, Basingstoke, UK).

PCR amplification and DNA sequencing

PCR and DNA sequencing primers were designed to target consensus sequences chosen after multiple nucleotide alignment of extended-spectrum ß-lactamase (CMY-1, FOX-2, FOX-3, MOX-1) genes using the Primer Calculator program (Williamstone Enterprises, Waltham, MA, USA). Primers (20 nucleotides) were CMYF1, starting at position 1 (FigureGo), and CMYR1, starting at position 1478 (FigureGo). Primers C1, C2, C3 and C4, PCR amplification and DNA sequencing have been described previously.5 The expected sizes of PCR products are 1460 bp (CMYF1/CMYR1), 847 bp (C1/C2) and 520 bp (C3/C4).



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Figure. Complete nucleotide sequence and predicted amino acid sequence of the blaCMY-11 ß-lactamase gene. The primers used to sequence the gene are underlined. Arrows indicate the direction of DNA sequencing. The stop codon is indicated with an asterisk. The ß-lactamase active site (SXXK), the typical class C motif (YXN) and the conserved triad (KTG) are heavily underlined. The putative signal peptide is shown with a dotted arrow. Possible promoter sequences (–35 and –10) and a ribosome-binding site (RBS) can be found upstream of the start codon. A terminal hairpin following the stop codon is marked by a solid arrow. The I-18 sequence is boxed. The differences in nucleotide and amino acid sequences from blaCMY-10 and CMY-10 are shaded.

 
DNA and protein sequence analysis

DNA sequence analysis was carried out with DNASIS for Windows (Hitachi Software Engineering America, San Bruno, CA, USA). Database searches for nucleotide and deduced amino acid sequence similarities were carried out at the NCBI website (http://www.ncbi.nlm.nih.gov).

Nucleotide sequence accession number

The blaCMY-11 sequence is filed in the GenBank database, accession number AF357600.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Properties of E. coli K983802.1

E. coli K983802.1 was isolated in 1998 from a 57-year-old female hospitalized in a neurology unit of the Kosin Medical Center and prescribed cefoxitin and ceftazidime. The isolate is distinguished by high-level resistance to cefalothin (MIC > 256 mg/L), cefoxitin (MIC > 256 mg/L), cefotetan (MIC = 256 mg/L), cefamandole (MIC = 256 mg/L), ceftazidime (MIC = 256 mg/L), co-amoxiclav (MIC = 64 mg/L) and aztreonam (MIC = 128 mg/L). This ß-lactam-resistance phenotype is similar to that of all strains of E. aerogenes (and E. cloacae) that overproduce chromosomal ß-lactamases, to E. aerogenes K9911729, which produces ß-lactamase CMY-10 and to K. pneumoniae CHO (pMVP-1), which produces ß-lactamase CMY-1.6

The plasmid content of E. coli K983802.1 was analysed by FIGE and a large plasmid (130 kb) was detected (data not shown). The strain was mated with E. coli J53AziR, and J53AziR transconjugants resistant to cefoxitin (20 mg/L) were recovered at a frequency of 4 x 10–5. All transconju-gants tested had one large plasmid (130 kb), designated pYMG-2, and produced a -lactamase with a pI value of 8.0, the same as those of CMY-1 and CMY-10 (data not shown). PCR amplification, with pYMG-2 DNA as template and primer pairs targeted to blaCMY-1-like and blaCMY-1 flanking sequences, yielded fragments of 1478 bp (CMYF1/ CMYR1), 847 bp (C1/C2) and 520 bp (C3/C4). The largest PCR product, obtained with primer pair CMYF1/CMYR1, was c. 18 bp longer than expected, as judged from blaCMY-1 and associated sequences.6

Sequence and phylogenetic analysis of blaCMY-11

The three PCR products were sequenced, yielding the complete nucleotide sequence of the ß-lactamase gene of pYMG-2. The gene comprises 1149 nucleotides encoding a predicted peptide of 382 amino acids (FigureGo). The deduced amino acid sequence has (1) the obligatory serine-active site ß-lactamase catalytic motif SXXK (Ser-ValSer-Lys, position 88–91 of the pre-processed peptide); (2) the class C ß-lactamase motif YXN; and (3) the KTG motif (FigureGo). GenBank, EMBL and DDBJ database searches (BLASTN) revealed that this ß-lactamase gene is most similar to blaCMY-10, blaCMY-1, blaCMY-8 and blaCMY-9 (99.9, 99.8, 97.9 and 97.8% homology, respectively), differing from blaCMY-10 by a single point mutation, T->G at position 944 (1197 in FigureGo). It has been designated blaCMY-11.

A multiple sequence alignment of the deduced amino acid sequence of CMY-11 with those of other class C ß-lactamases revealed that it is most closely related to CMY-10 (99.7%) and CMY-1 (99.5%). Compared with CMY-1, CMY-10 has a single amino acid substitution (Asn366Ile), whereas CMY-11 has two (Ile315Ser, Asn 366Ile) (TableGo). CMY-1, CMY-10 and CMY-11 were all found in Korean clinical isolates. A likely evolutionary sequence is CMY-1->CMY-10->CMY-11. The Asn366Ile substitution found in CMY-10 and CMY-11 is common to CMY-8, CMY-9, FOX-1, FOX-2, FOX-3, FOX-4, FOX-5, MOX-1, MOX-2 and ACC-1. The Ile315Ser change in CMY-11 is also found in FOX-1 to FOX-5.


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Table. Nucleotide and amino acid differences between blaCMY-1, blaCMY-8, blaCMY-9, blaCMY-10 and blaCMY-11
 
Characteristics such as substrate preference and inhibition profile are less discriminatory for class C ß-lactamases than for class A enzymes. Sub-classification according to amino acid sequence is more informative. A phylogenetic tree of 62 AmpC genes can be found at The Hall Laboratory website (http://www.rochester.edu/College/BIO/labs/HallLab/AmpC_Phylo.html ). Since CMY-11 differs from CMY-1 by only two amino acids, it can be expected to cluster with CMY-1, CMY-8, CMY-9 and MOX-1.

Genetic context of blaCMY-11

The sequence preceding blaCMY-11, nucleotides 1–253 (FigureGo), less an 18 nucleotide duplication (I-18, TTTATACTTCCTATACCC; nucleotides 72–89; FigureGo and TableGo), is identical to that upstream from blaCMY-1.6 All putative gene expression signals are preserved. The truncated sequence preceding the 18 nucleotide duplication, I-18 (nucleotides 1–71, FigureGo), has been identified as part of the unusual class 1 integrons In6 and In77 and the integron-like structure on pSAL-1.8 In all cases the homology is to a sequence in the modified 3'-conserved sequence of these integrons and is lost abruptly after the I-18 sequence,8 duplicated or not. This finding indicates insertion at this point (I-18) of different sequences, encoding a dihydrofolate reductase (dhfrX, In7),7 a chloramphenicol acetyl transferase (cat, In6)7 and various ampC genes (blaDHA-1, pSAL-1; blaCMY-1, pMVP-1; blaMOX-1, pRMOX-1; blaCMY-11, pYMG-2),8 indicating that this is a secondary locus for gene capture by integrons such as In6 and In7. Although the variety of sequences that have been inserted, apparently at the same point, is reminiscent of the different gene cassettes found in the standard variable insert region of integrons,9,10 there is no evidence that 59 base elements are needed for insertion at the secondary site. This would indicate involvement of another site-specific, integron-associated recombination system that is able to capture antibiotic-resistance genes on to mobile DNA structures and so promote their dissemination among bacteria of clinical importance.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank Dr Y. B. Kim for helpful suggestions and Dr J. Kim for the generous gift of E. coli J53 AziR. This work was supported by a research grant from the Korea Science and Engineering Foundation (1999-2-211-002-5) and by a Youngdong University research grant (YU-01-15) of 9th Industry University Institute Cooperative Technology Development Consortium in Chungbuk (2001).


    Notes
 
* Corresponding author. Tel: +82-43-740-1112; Fax: +82-43-740-1109; E-mail: sanghee{at}youngdong.ac.kr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Chanal, C., Sirot, D., Romaszko, J. P. & Sirot, J. (1996). Survey of prevalence of extended-spectrum ß-lactamases among Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 38, 127–32.[Abstract]

2 . Ambler, R. P. (1980). The structure of ß-lactamases. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences 289, 321–31.[ISI][Medline]

3 . Marchese, A., Arlet, G., Schito, G. C., Lagrange, P. H. & Philippon, A. (1998). Characterization of FOX-3, an AmpC-type plasmid-mediated ß-lactamase from an Italian isolate of Klebsiella oxytoca . Antimicrobial Agents and Chemotherapy 42, 464–7.[Abstract/Free Full Text]

4 . Ford, N., Nolan, C., Ferguson, M. & Ockler, M. (1989). Rapid disruption of bacterial colonies to test the size of plasmids and transfer of DNA from agarose to solid supports. In Molecular Cloning: A Laboratory Manual, 2nd edn, (Sambrook, J., Fritsch, E. F. & Maniatis, T., Eds), pp. 1.32, 9.34–7. Cold Spring Harbor Laboratory Press, New York, NY.

5 . Lee, S. H., Kim, J. Y., Shin, S. H., Lee, S. K., Choi, M. M., Lee, I. Y. et al. (2001). Restriction fragment length dimorphism-PCR method for the detection of extended-spectrum beta-lactamases unrelated to TEM- and SHV-types. FEMS Microbiology Letters 200, 157–61.[ISI][Medline]

6 . Bauernfeind, A., Stemplinger, I., Jungwirth, R., Wilhelm, R. & Chong, Y. (1996). Comparative characterization of the cephamycinase blaCMY-1 gene and its relationship with other ß-lactamase genes. Antimicrobial Agents and Chemotherapy 40, 1926–30.[Abstract]

7 . Stokes, H. W., Tomaras, C., Parsons, Y. & Hall, R. M. (1993). The partial 3'-conserved segment duplications in the integrons In6 from pSa and In7 from pDGO100 have a common origin. Plasmid 30, 39–50.[ISI][Medline]

8 . Verdet, C., Arlet, G., Barnaud, G., Lagrange, P. H. & Philippon, A. (2000). A novel integron in Salmonella enterica serovar Enteritidis, carrying the bla (DHA-1) gene and its regulator gene ampR, originated from Morganella morganii. Antimicrobial Agents and Chemotherapy 44, 222–5.[Abstract/Free Full Text]

9 . Hall, R. M. & Collis, C. M. (1995). Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Molecular Microbiology 15, 593–600.[ISI][Medline]

10 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 3015–27.[ISI][Medline]

Received 30 April 2001; returned 10 August 2001; revised 28 August 2001; accepted 7 November 2001