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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 MuellerHinton 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 310) 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 (Figure), and CMYR1, starting at position 1478 (Figure
). 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).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 105. 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 (Figure). The deduced amino acid sequence has (1) the obligatory serine-active site ß-lactamase catalytic motif SXXK (Ser-ValSer-Lys, position 8891 of the pre-processed peptide); (2) the class C ß-lactamase motif YXN; and (3) the KTG motif (Figure
). 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 Figure
). 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) (Table). 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.
|
Genetic context of blaCMY-11
The sequence preceding blaCMY-11, nucleotides 1253 (Figure), less an 18 nucleotide duplication (I-18, TTTATACTTCCTATACCC; nucleotides 7289; Figure
and Table
), 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 171, Figure
), 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Ambler, R. P. (1980). The structure of ß-lactamases. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences 289, 32131.[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, 4647.
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, 9.347. 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, 15761.[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, 192630.[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, 3950.[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, 2225.
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, 593600.[ISI][Medline]
10 . Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 301527.[ISI][Medline]
Received 30 April 2001; returned 10 August 2001; revised 28 August 2001; accepted 7 November 2001