Plasmid-mediated and inducible cephalosporinase DHA-2 from Klebsiella pneumoniae

Nicolas Fortineau, Laurent Poirel and Patrice Nordmann*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A Klebsiella pneumoniae strain resistant to cefoxitin and oxyimino-cephalosporins was cultured from a child hospitalized in Paris, France, in 1992. This isolate harboured a ß-lactamase gene located on an approximately 200 kb non-self-transferable plasmid. The ß-lactamase identified, DHA-2, shared 99% amino acid identity with the AmpC enzyme of Morganella morganii. DHA-2 was a point-mutant derivative of DHA-1 identified previously in a Salmonella enteritidis isolate. DHA-2 expression was inducible due to an ampR regulatory gene. This is the first report of an inducible and plasmid-located cephalosporinase from K. pneumoniae.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Seventeen plasmid-mediated AmpC cephalosporinases have been reported so far among enterobacterial isolates since the discovery of MIR-1 cephalosporinase in 1990.1,2 These ß-lactamases are structurally related to the inducible chromosomally mediated AmpC enzymes. CMY-2, LAT-1, LAT-2 and LAT-3 are related to AmpC of Citrobacter freundii, MIR-1 and ACT-1 to AmpC of Enterobacter cloacae, DHA-1 to AmpC of Morganella morganii, ACC-1 to AmpC of Hafnia alvei, whereas the phylogeny of other enzymes (CMY-1, MOX-1, FOX-1, FOX-2 and FOX-3) remains unclear.16 Compared with the plasmid-mediated class A extended-spectrum ß-lactamases, these plasmid-encoded cephalosporinases are active against cephamycins (except ACC-1), and against oxyimino-cephalosporins. Their in vitro activity is not inhibited by clavulanate.

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.


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

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{alpha} (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{alpha} 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{alpha} 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 3.5–9.5) 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{alpha} 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 715–735, 340–359 and 92–111, respectively) and the 5'-end of the ampC gene (5'-GGCTTTGACTCTTTCGGTATTC-3') (position 2737–2758) were used. A primer pair made of the 3'-end of blaDHA-1 (5'-TTCTGCCGCTGATAATGTCGC-3') (position 1711–1731) 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 4551–4572 and 5801–5821, 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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A routine susceptibility test on K. pneumoniae 45956 revealed an unusual antagonism between imipenem or cefoxitin discs and ceftazidime discs. Despite repeated conjugation experiment failures, E. coli DH5{alpha} electroporants were obtained after selection on plates containing ticarcillin, with plasmid DNA extracted from K. pneumoniae 45956. As found for K. pneumoniae 45956, ceftazidime resistance was not inhibited by clavulanate and was associated with cefoxitin resistance (TableGo). Susceptibility testing by disc diffusion did not detect other antibiotic resistance markers in this recombinant E. coli DH5{alpha} strain.


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Table. MICs of ß-lactams for clinical isolate K. pneumoniae 45956, electroporant E. coli DH5{alpha}(pFOR-1) and reference strain E. coli DH5{alpha}
 
IEF analysis showed that the E. coli DH5{alpha} electroporant expressed only one ß-lactamase with a pI value of 7.8, while K. pneumoniae 45956 had an additional ß-lactamase with a pI value of 7.6, which probably corresponded to the chromosomally mediated SHV-1 type ß-lactamase. Plasmid DNA analysis of the E. coli DH5{alpha} electroporant identified an approximately 200 kb plasmid, pFOR-1, which was also present in K. pneumoniae 45956 (data not shown). The ß-lactamase specific activity of E. coli DH5{alpha}(pFOR-1) on cephalothin was increased by 6.4- and 5.4-fold when imipenem and cefoxitin were added, respectively, as ß-lactam inducer (basal level 1 U/mg of protein).

Using a set of primers specific for the blaDHA-1 gene and E. coli DH5{alpha}(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 (FigureGo). 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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Figure. Comparison of the deduced amino acid sequences of (a) the ß-lactamase DHA-2 from K. pneumoniae 45956 with that of DHA-1 from S. enteritidis1 (underlined amino acids are those that may be involved in the catalytic site of these AmpC enzymes); and (b) the AmpR proteins of K. pneumoniae 45956 (Kp) with that of M. morganii strain 17 (Mm).

 
Since DHA-1 was found to be inducible due to the presence of a reverse transcribed ampR gene, primers located in the 3' ends of this ampR gene and in the blaDHA-2 gene allowed amplification and sequencing of a 2044 bp fragment. The deduced AmpR protein encoded by plasmid pFOR-1 had 98% amino acid identity with the AmpR of M. morganii strain 17 (FigureGo). The inter-cistronic region of the ampC and ampR genes, which contained the promoter sequences for ampC and ampR expression, was very similar to that found either in the S. enteritidis plasmid that contained blaDHA-1 or in M. morganii.1,7 Only 1 bp mutation was found near the –35 sequence of the promoter region of ampR. Since the DNA region surrounding the ampC and ampR genes is conserved in M. morganii,7 PCR was used to search for the presence of hybF, hybE and orf1 genes. orf1 was not identified in plasmid pFOR-1 while hybF- and hybE-like genes were found downstream of the ampR gene. Sequence analysis of the PCR fragments obtained revealed that the deduced protein HybF differed by only one amino acid from HybF of M. morganii strain 1,7 and by only two amino acid substitutions out of the 62 amino acids deduced from the partial sequence of the gene. These results further indicated the M. morganii chromosomal origin of blaDHA-2.

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.


    Acknowledgments
 
We are grateful to Patrick Berche in whose laboratory K. pneumoniae 45956 was isolated and to Thierry Naas for critical review of the manuscript. This work was funded by the Ministère de la Recherche, Université Paris XI, Faculté de Médecine Paris Sud (grant UPRES, JE-2227).


    Notes
 
* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Barnaud, G., Arlet, G., Verdet, C., Gaillot, O., Lagrange, P. H. & Philippon, A. (1998). Salmonella enteritidis: AmpC plasmidmediated inducible ß-lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrobial Agents and Chemotherapy 42, 2352–8.[Abstract/Free Full Text]

2 . Barnaud, G., Arlet, G., Danglot, C. & Philippon, A. (1997). Cloning and sequencing of the gene encoding the AmpC ß-lactamase of Morganella morganii. FEMS Microbiology Letters 148, 15–20.[ISI][Medline]

3 . Bauernfeind, A., Schneider, I., Jungwirth, R., Sahly, H. & Ullmann, U. (1999). A novel type of AmpC ß-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. Antimicrobial Agents and Chemotherapy 43, 1924–31.[Abstract/Free Full Text]

4 . Medeiros, A. A. (1997). Evolution and dissemination of ß-lactamases accelerated by generations of ß-lactam antibiotics. Clinical Infectious Diseases 24, 19S–45S.

5 . Girlich, D., Naas, T., Bellais, S., Poirel, L., Karim, A. & Nordmann, P. (2000). Biochemical-genetic characterization, and regulation of expression of an ACC-1-like chromosome-borne cephalosporinase from Hafnia alvei. Antimicrobial Agents and Chemotherapy 44, 1470–8.[Abstract/Free Full Text]

6 . Papanicolaou, G. A., Medeiros, A. A. & Jacoby, G. A. (1990). Novel plasmid-mediated ß-lactamase (MIR-1) conferring resistance to oxyimino- and {alpha}-methoxy ß-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 34, 2200–9.[ISI][Medline]

7 . Poirel, L., Guibert, M., Girlich, D., Naas, T. & Nordmann, P. (1999). Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates. Antimicrobial Agents and Chemotherapy 43, 769–76.[Abstract/Free Full Text]

8 . 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, 222–5.[Abstract/Free Full Text]

9 . Poirel, L., Naas, T., Guibert, M., Chaibi, E. B., Labia, R. & Nordmann, P. (1999). Molecular and biochemical characterization of VEB-1, a novel class A extended spectrum ß-lactamase encoded by an Escherichia coli integron gene. Antimicrobial Agents and Chemotherapy 43, 573–81.[Abstract/Free Full Text]

10 . Verdet, C., Arlet, G., Ben Redjeb, S., Ben Hassen, A., Lagrange, P. H. & Philippon, A. (1998). Characterization of CMY-4, an AmpC-type plasmid-mediated ß-lactamase in a Tunisian clinical isolate of Proteus mirabilis. FEMS Microbiology Letters 169, 235–40.[ISI][Medline]

Received 2 May 2000; returned 19 August 2000; revised 14 September 2000; accepted 18 October 2000