Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France
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Abstract |
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Introduction |
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The only known plasmid-encoded and inducible cephalosporinase is DHA-1 from Salmonella enteritidis.1 The blaDHA-1 gene is closely related to the chromosomal M. morganii ampC gene and is associated on the same plasmid with the regulator ampR gene, which is responsible for inducibility.1,7 Recently, these genes were found to be located on a peculiar class 1 integron but not as a mobilizable cassette.8
Routine susceptibility testing of a Klebsiella pneumoniae clinical isolate revealed an inducible ceftazidime resist-ance phenotype. Thus, we proceeded to the analysis of its ß-lactamase content.
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Materials and methods |
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K. pneumoniae 45956 was isolated from a perianal abscess of a 6-year-old child at the Hôpital Necker (Paris, France) in 1992. It was identified by the API-50 CH system (bioMérieux, Marcy-l'Étoile, France). Rifampicin-resistant Escherichia coli JM1099 and E. coli DH5 (Gibco-BRL LifeTechnologies, Eragny, France) were used as recipient strains for conjugation and electroporation experiments, respectively. E. coli strain NCTC 50192 carrying plasmids of 154, 66, 38 and 7 kb was used as a control in plasmidsizing studies.9
Antimicrobial agents and MIC determination
The antimicrobial agents and their sources have been described elsewhere.9 A disc diffusion assay was used for routine detection of antibiotic susceptibility on Mueller Hinton (MH) agar plates (Sanofi-Diagnostics Pasteur, Marnes-La-Coquette, France). MICs were determined by an agar dilution technique on MH agar plates as reported.9
Plasmid content and mating-out assays
Plasmid DNAs of K. pneumoniae and E. coli DH5 electroporants were extracted using the Qiagen plasmid DNA maxi kit (Qiagen, Courtaboeuf, France) and analysed as described.9 Direct transfer of the ceftazidime resistance marker into E. coli JM109 was attempted by liquid and solid mating-out assays at 37°C.9 Transconjugant selection was performed on trypticase soy agar plates (SanofiDiagnostics Pasteur) containing ceftazidime (4 mg/L) and rifampicin (150 mg/L). Electrotransformation of the plasmid DNA suspension of K. pneumoniae 45956 into E. coli DH5
was performed with ticarcillin (100 mg/L) selection.
Isoelectric focusing and ß-lactamase induction assays
ß-Lactamase extracts of K. pneumoniae 45956 and of one E. coli electroporant were subjected to analytical isoelectric focusing (IEF) on an ampholine polyacrylamide gel (pH range 3.59.5) on a flatbed apparatus (FBE-3000; Amersham Pharmacia Biotech, Orsay, France) as described previously.9 ß-Lactamase basal and induced levels were determined as described7 with cultures of electroporant E. coli DH5 harbouring the natural plasmid pFOR-1 from K. pneumoniae 45956. Induction of ß-lactamase was performed with imipenem (0.5 mg/L) and cefoxitin (5 mg/L), respectively. The specific ß-lactamase activities were determined with 100 µM cephalothin as substrate.7 One unit of enzyme activity was defined as the activity that hydrolysed 1 µmol of cephalothin per min. The total protein content was measured with the Bio-Rad DC Protein assay kit (Bio-Rad, Ivry sur Seine, France).
PCR experiments, DNA sequencing and protein analysis
PCR experiments were performed using plasmid pFOR-1 as a template and sets of primers internal to several ampC genes (E. coli, E. cloacae, C. freundii, Serratia marcescens, H. alvei and M. morganii).5,7,10 Since the set of primers corresponding to the blaDHA-1 chromosomally mediated cephalosporinase of M. morganii (GenBank accession number AF055067) gave a positive result, internal primers located in M. morganii putative ampR (5'-GTTTCCGTACGGGACTGTAAC-3'), hybF (5'-TGAGTGCGGCGGACATTATC-3') or hybE (5'-TTGCCGTACGGCATCATGAC-3') genes (positions 715735, 340359 and 92111, respectively) and the 5'-end of the ampC gene (5'-GGCTTTGACTCTTTCGGTATTC-3') (position 27372758) were used. A primer pair made of the 3'-end of blaDHA-1 (5'-TTCTGCCGCTGATAATGTCGC-3') (position 17111731) and (5'-ACCACCACAAAGCGCGAGTC-3') (position 4822 4841) in orf1 was also used to determine the genetic environment of the cephalosporinase gene. Furthermore, the plasmid-mediated cephalosporinase genes blaDHA-1, blaCMY-1 and blaMOX-1 neighboured a region that is shared by integrons In6 and In7.8 Thus, PCR experiments were also attempted using primer 5'-GTGGTTTATACTTCCTATACCC-3' located in this common region and one in blaDHA-1 (positions 45514572 and 58015821, respectively, of the nucleotide sequence in GenBank accession number AJ237702), as well as class 1 integron-specific primers located either in the 5'-CS or in the 3'-CS and in blaDHA-1.
The PCR fragments obtained were sequenced on both strands (ABI 377; P.E. Biosystems, Les Ulis, France). The nucleotide and the deduced protein sequences were analysed online at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). Multiple nucleotide and protein sequence alignments were carried out online using the program Clustal_W available at the University of Cambridge website (http://www2.cbi.ac.uk/clustalW).
Nucleotide sequence accession number
The nucleotide sequence reported in this paper will appear under GenBank accession number AF259520.
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Results and discussion |
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Using a set of primers specific for the blaDHA-1 gene and E. coli DH5(pFOR-1) DNA as template, a 1048 bp PCR fragment was obtained. Sequencing revealed a deduced protein, named DHA-2, with 98% amino acid identity with the plasmid-mediated cephalosporinase DHA-1 from S. enteritidis and with the chromosomally borne cephalosporinase of M. morganii (Figure
). The amino acid changes in DHA-2 as compared with DHA-1 were not located in the putative active site of AmpC enzymes.7
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The neighbouring regions located at the 5' end of blaDHA-1 found in several class 1 integrons and around the plasmid-mediated cephalosporinase genes, as identified by Verdet et al.,8 were not found in the blaDHA-2 environment. However, it is not possible to rule out PCR failure due to point mutations in this DNA region leading to lack of annealing of the designed primers. Additionally, PCR experiments using 5'-CS and 3'-CS specific primers of class 1 integrons failed to show the blaDHA-2 location inside a typical class 1 integron.
This work identified the second plasmid-mediated cephalosporinase that is a derivative of the chromosomally encoded AmpC of M. morganii. This is the first case of an inducible cephalosporinase from a European clinical isolate. Unlike S. enteritidis, K. pneumoniae is a nosocomial species of clinical relevance. As with clavulanate-inhibited extended-spectrum ß-lactamases, this report provides additional evidence for K. pneumoniae being the main reservoir of plasmid-mediated cephalosporinases. However, it remains puzzling that the ampC-derived genes of M. morganii, once plasmid located, are associated with ampR genes as opposed to the other known plasmid-mediated cephalosporinase genes. The mechanisms of gene excision and insertion into the plasmid are perhaps different and M. morganii may simply favour large-size DNA fragment excisions.
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Acknowledgments |
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Notes |
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References |
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Verdet, C., Arlet, G., Barnaud, G., Lagrange, P. H. & Philippon, A. (2000). A novel integron in Salmonella enterica serovar enteriditis, carrying the blaDHA-1 gene and its regulator gene ampR, originated from Morganella morganii. Antimicrobial Agents and Chemotherapy 44, 2225.
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Received 2 May 2000; returned 19 August 2000; revised 14 September 2000; accepted 18 October 2000