Characterization of CMY-type ß-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area

Dominique Decré1,*, Charlotte Verdet2, Laurent Raskine3, Hervé Blanchard4, Béatrice Burghoffer1, Alain Philippon4, Marie José Sanson-Le-Pors2, Jean Claude Petit1 and Guillaume Arlet3

1 Service de Bactériologie, Hôpital Saint Antoine,UFR Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012 Paris; 2 Service de Bactériologie, Hôpital Tenon, UFR Saint-Antoine, Paris; 3 Service de Bactériologie Hôpital Lariboisière; 4 Service de Bactériologie, CHU Cochin, AP-HP, Paris, France

Received 13 November 2001; returned 20 March 2002; revised 1 May 2002; accepted 2 August 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with ß-lactam resistance phenotypes consistent with production of an AmpC-type ß-lactamase. The predicted amino acid sequences of the enzymes were typical of class C ß-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant ß-lactamase, CMY-12. The AmpC ß-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type ß-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Transferable resistance to extended-spectrum cephalosporins and monobactams in Enterobacteriaceae was first described in the early 1980s.1,2 These enzymes evolved by multiple point mutations in the genes encoding the common plasmid-mediated TEM-1, TEM-2 and SHV-1 class A enzymes. A number of other extended-spectrum ß-lactamase genes, including those for BIL, CMY, LAT and MOX ß-lactamases, have been reported. Nucleotide sequence analyses have shown that some of these families are closely related to the AmpC-type chromosomal ß-lactamases of Citrobacter freundii (CMY-2,3,4 CMY-3,5 CMY-4,6 BIL-1,7 LAT-1,8,9 LAT-210), Enterobacter cloacae (MIR-1,11,12 ACT-113), Morganella morganii (DHA-1,14,15 DHA-216) and Hafnia alvei (ACC-117,18), whereas the phylogeny of others (CMY-1 types, FOX-types and MOX-1) is unclear or unknown.

Some of the newly acquired ampC genes are located on plasmids that transfer non-inducible cephalosporin resistance to Klebsiella pneumoniae,3,9,12,13,1922 Escherichia coli,7,17,2224 Proteus mirabilis,6,17,22,23 Klebsiella oxytoca,22,25 Enterobacter aerogenes10 and Salmonella sp.4,15,22,26 In addition, the first example of a chromosome-encoded AmpC ß-lactamase (CMY-3) in P. mirabilis was reported recently.5

In this study we characterized plasmid- and chromosome-encoded CMY-type ß-lactamases from three clinical isolates of P. mirabilis and two clinical isolates of K. pneumoniae from Paris hospitals, and we report a new CMY ß-lactamase variant.


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

The characteristics of the clinical strains studied are summar-ized in Table 1. These strains are resistant to the combination of amoxicillin with clavulanic acid (co-amoxiclav) and to cefoxitin. Two previously described CMY-type ß-lactamase-producing strains were used as controls: P. mirabilis H223b (CMY-4)6 and Salmonella senftenberg (CMY-2).4


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Table 1. Origins of the P. mirabilis and K. pneumoniae isolates investigated
 
The rifampicin-resistant strain of E. coli K12 J53-2 (rifR) was used as recipient in transfer experiments.

Antibiotic susceptibility and synergy testing

MICs were determined for several ß-lactams by the standard agar dilution method with Mueller–Hinton agar (Bio-Rad, Marnes la Coquette, France). Inocula (104–105 cfu per spot) were delivered with a multipoint inoculator. Resistance to other antibiotics, such as aminoglycosides and quinolones, was determined by the disc diffusion method on Mueller–Hinton agar, according to the recommendations of the Comité de l’Antibiogramme de la Société Française de Microbiologie.27 Class A extended-spectrum ß-lactamases were detected by a synergy test using cefepime and co-amoxiclav.27

Double-disc synergy tests were performed as described15 with various cephalosporins: cefoxitin, cefotetan, ceftazidime and cefotaxime and RO48-1220 (20 µg per disc) (Hoffman-La Roche Ltd, Basel, Switzerland), which is a ß-lactamase inhibitor that protects expanded-spectrum cephalosporins against strains producing group 1 and group 2be enzymes.2 The disc with inhibitor was placed on the agar surface, 30 mm away from the central disc.15

ß-Lactamase analysis

Analytical isoelectric focusing (IEF) was performed in ampholine polyacrylamide gels (pH 3.5–10) (Pharmacia-Biotech, Saclay, France) for 18 h at 400 V, using crude cell-free sonic extracts and a Multiphor apparatus (Pharmacia-Biotech). ß-Lactamase activity was detected by the nitrocefin procedure.28 The following ß-lactamases (plasmid, pI) were used as standards: TEM-1 (pIP1100, pI 5.4), TEM-2 (RP4, pI 5.6), TEM-3 (pCFF04, pI 6.3), OXA-1 (RGN238, pI 7.4), SHV-4 (pUD21, pI 7.8) and CMY-2b (pSENF, pI 9.0).

PCR amplification and molecular characterization of TEM-type ß-lactamases

Primers OT3 and OT4 were used to amplify putative blaTEM genes, which were characterized by PCR–RFLP analysis, as described previously.29 PCR conditions were as follows: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C and a final extension step of 7 min at 72°C. All enzymes used were purchased from Roche Molecular Biochemicals (Meylan, France) or New England Biolabs (Ozyme, Saint Quentin en Yvelynes, France).

Sequences of class C ß-lactamases

Parts of the putative class C ß-lactamase genes of the three strains of P. mirabilis and the two strains of K. pneumoniae were amplified using degenerate oligonucleotide primers (ampC A1, ampC A2) based on the consensus sequence of the ampC genes of E. coli, E. cloacae and C. freundii. The sequences of primers were as follows: ampC A1 (5'-GGAATTCCTWTGCTGCGCBCTGCTGCT-3') and ampC A2 (5'-CGGGATCCCTGCCAGTTTTGATAAAA-3').4,6 The resulting PCR products were sequenced, as described by Sanger et al.,30 using oligonucleotide primers, fluorescently labelled dideoxynucleotides, Taq polymerase and an ABI 373A DNA sequencer (PE Applied Biosystems, Foster City, CA, USA).

The DNA sequences encoding the intact mature proteins were then amplified with the consensus primers for blaBIL-1, blaLAT-1, blaCMY-2 and the ampC gene of C. freundii OS60 (ampC1, 5'-ATGATGAAAAAATCGTTATGC-3'; ampC2, 5'-TTGCAGCTTTTCAAGAATGCGC-3'), and the products were sequenced as above.4,6 The products of two separate PCRs were sequenced on both strands.

The BLAST and FASTA programs were used to search databases for similar nucleotide and amino acid sequences. The Clustal X program was used to align multiple protein sequences.3133

Conjugation

Mating experiments were performed by mixing equal volumes (1 mL) of exponentially growing cultures of each test strain and E. coli K12 J53-2 in brain–heart infusion broth (Difco, Detroit, MI, USA) and incubating the mixture for 3 h at 37°C. ß-Lactam-resistant E. coli J53-2 transconjugants were selected on Drigalski agar (Bio-Rad) containing rifampicin (256 mg/L) and cefoxitin (8 or 16 mg/L).

Plasmid analysis

Plasmid DNA was extracted from the clinical isolates of P. mirabilis and K. pneumoniae, from E. coli transconjugants and from the two control strains by alkaline lysis, as described.34 RP4 (54 kb), pCFF04 (85 kb) and pIP173 (126.8 kb) were used as reference plasmids.

PFGE analysis

Genomic DNA, prepared as described35 and digested with XbaI (Roche Molecular Biochemicals), was subjected to PFGE in a CHEF DRIII device (Bio-Rad). DNA fragments were separated in a 1% (w/v) agarose gel in 0.5x Tris–Borate–EDTA buffer at 200 V for 20 h, with pulse times ranging from 5 to 30 s.

Genomic fingerprinting by repetitive-element PCR (rep-PCR) analysis

rep-PCR was performed as described.35 The primers used were REP-1R-Dt (IIINCGNCGNATCNGGC) and REP2-Dt (NCGNCTTATCNGGCCTAC).36 The reaction was carried out in a GeneAmp PCR system 9700 (PE Applied Biosystems). Amplified DNA was analysed by agarose gel electrophoresis.

Hybridization studies

Hybridization was carried out after transfer to a nylon membrane (Hybond N, Pharmacia-Biotech) of both plasmid DNA and XbaI restriction fragments of total DNA separated by PFGE. The probe consisted of an internal DNA fragment (450 bp) from the CMY-4 ampC ß-lactamase gene, obtained by PCR with degenerate primers (ampC A1 and ampC A2) and labelled with [{alpha}-32P]dATP.6

Nucleotide sequence accession numbers

The GenBank accession numbers of the DNA sequences are: P. mirabilis 22317 No. Y16783; P. mirabilis PLAR No. Y16782; P. mirabilis 34955 No. Y16785; K. pneumoniae 169 No. Y16784; K. pneumoniae 9701 No. Y16781.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antibiotic susceptibility and ß-lactamase analysis

All clinical isolates were highly resistant to penicillins (amoxicillin, ticarcillin, piperacillin); clavulanic acid did not significantly restore susceptibility to amoxicillin or ticarcillin (Table 2). As reported by others, tazobactam partially restored susceptibility to piperacillin.4,6,26,37 All of the strains were susceptible to moxalactam, cefepime and imipenem, and no synergy was observed between cefepime and co-amoxiclav. Synergy was observed between the inhibitor RO48-1220, which is active against class C ß-lactamases, and the four cephalosporins tested with all of the clinical isolates (data not shown). These results suggest that each of the strains produces an AmpC-type ß-lactamase. Since 1990, several plasmid-mediated AmpC-type ß-lactamases have been characterized from Enterobacteriaceae lacking inducible AmpC enzymes, such as K. pneumoniae,3,9,12,13,1922 P. mirabilis 6,17,22,23 and Salmonella sp.,4,15,22,26 and in naturally occurring AmpC species, such as E. coli 7,17,2224 and E. aerogenes.10


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Table 2. MICs (mg/L) of ß-lactams for P. mirabilis 22317, PLAR and 34955 and for K. pneumoniae 169 and 9701 and their E. coli transconjugants (Tc)
 
IEF analysis revealed the presence of ß-lactamases with pIs > 9.0 in P. mirabilis 22317, PLAR and 34955, and K. pneumoniae 169 and 9701 (data not shown). These values are consistent with production of AmpC-type ß-lactamases. In addition, ß-lactamases with pIs of 7.7 were detected in the two K. pneumoniae strains (169 and 9701). These enzymes are probably related to the SHV-1 chromosomal ß-lactamase commonly found in this species. ß-Lactamases with pIs of 5.4 were detected in P. mirabilis PLAR, P. mirabilis 22317 and K. pneumoniae 9701, and were shown to be TEM-1 by PCR–RFLP (data not shown). Finally, a ß-lactamase with a pI of 5.6 detected in P. mirabilis 34955 was shown to be TEM-2 by PCR–RFLP (data not shown).

Characterization of AmpC-type ß-lactamases

PCR experiments using the degenerate primers ampC A1 and ampC A2 confirmed that each of the five strains investigated produces an AmpC-type ß-lactamase. The predicted amino acid sequences show that each enzyme has the ß-lactamase active site SVSK, the conserved KTG triad and the typical class C YXN motif. Analysis of the partial sequences showed that all five enzymes are related to the AmpC ß-lactamase of C. freundii.

Transferable class C ß-lactamases have been found worldwide, most frequently in K. pneumoniae, but also in E. coli, P. mirabilis and salmonellae. CMY-2 is the most prevalent AmpC-like enzyme and has the widest geographical distribution, having been reported in Algeria, France, Germany, Greece, India, Pakistan, Taiwan, the UK and the USA,37,38 and recently in Spain.22 CMY-4 was first identified in a P. mirabilis isolate from Tunisia and is also believed to originate from the chromosome of C. freundii.6

Subsequent DNA amplifications and sequence analyses showed that the gene from K. pneumoniae 169 is 100% identical to blaCMY-2 and that the genes from P. mirabilis 22317, P. mirabilis PLAR and K. pneumoniae 9701 are 100% identical to blaCMY-4. Finally, the sequence of the gene from P. mirabilis 34955 was found to be highly related to the sequences of several plasmid-mediated cephalosporinase genes related to the C. freundii group (98–99% homology) (Table 3). This new cephalosporinase, encoded by the gene named CMY-12, is a member of the C. freundii group and differs from other members of the group by an A171S substitution.


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Table 3.  Differences (in bold type) in the amino acid sequences of the AmpC ß-lactamases of the C. freundii group
 
Transfer of resistance and plasmid analysis

E. coli J53-2 transconjugants were obtained with K. pneumoniae 169 and K. pneumoniae 9701, and the two control strains P. mirabilis H223b (CMY-4) and S. senftenberg (CMY-2) as donors. The susceptibility patterns of the transconjugants to ß-lactams were found to be similar to those of the donor strains. Resistance to aminoglycosides and sulphonamides was also transferred from K. pneumoniae 9701. In contrast, attempts to transfer ß-lactam resistance by conjugation from the three P. mirabilis isolates were unsuccessful.

The two K. pneumoniae strains and their E. coli J53-2 transconjugants harbour large plasmids (>130 kb). A smaller plasmid (50 kb) is present in P. mirabilis 34955 (Figure 1).



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Figure 1. (a) Plasmid analysis; (b) hybridization with the ampC probe. Lane 1, P. mirabilis 22317; lane 2, P. mirabilis H223b (CMY-4); lane 3, E. coli J53-2 Tc Pm H223b; lane 4, P. mirabilis PLAR; lane 5, P. mirabilis 34955; lane 6, RP4 (55 kb); lane 7, TEM-3 (85 kb); lane 8, pIP173 (126 kb); lane 9, K. pneumoniae 169; lane 10, E. coli J53-2 Tc Kp 169; lane 11, K. pneumoniae 9701; lane 12, E. coli J53-2 Tc Kp 9701; lane 13, S. senftenberg; lane 14, E. coli J53-2 Tc Ss.

 
The genes for the AmpC-type ß-lactamases have been located on plasmids of various sizes, from 7 to 180 kb.37 In contrast, it was reported recently that a P. mirabilis strain harbours a chromosomal copy of an ampC homologue.5

Hybridization studies

To determine the location of the ampC genes in the K. pneumoniae and P. mirabilis isolates, plasmid DNA and XbaI restriction fragments of total DNA separated by PFGE were hybridized with an ampC-specific probe. The probe hybridized strongly with plasmid DNA both from K. pneumoniae 169 and 9701 and from their E. coli J53-2 transconjugants, as well as with the plasmid DNA from the control strains and their E. coli tranconjugants (Figure 1). In contrast, the probe did not hybridize to plasmid DNA from the three strains of P. mirabilis; the low level of hybridization observed with plasmid preparations from these strains probably corresponds to chromosomal DNA present in the extracts.

In contrast, the ampC-specific probe hybridized strongly to large XbaI restriction fragments (>400 kb), separated by PFGE, from P. mirabilis 22317, PLAR and 34955, and to a much smaller fragment from the control strain, P. mirabilis H223b, consistent with the plasmid size described previously6 (Figure 2). These results suggest that the ampC genes of the three clinical isolates of P. mirabilis are chromosomal. Bret et al.5 suggested that the ampC gene could have integrated into the chromosome of P. mirabilis via a transposon. Further studies are underway to examine this hypothesis.



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Figure 2. (a) PFGE analysis of genomic DNA fragments digested with XbaI from four P. mirabilis isolates carrying the AmpC-type ß-lactamase (negative shown). (b) Hybridization with the ampC probe. Lane 1, P. mirabilis 22317; lane 2, P. mirabilis H223b (CMY-4); lane 3, P. mirabilis LAR; lane 4, P. mirabilis 34955.

 
Interestingly, P. mirabilis 22317 and P. mirabilis PLAR have similar PFGE profiles, suggesting a clonal origin. rep-PCR fingerprinting, which has been used for epidemiological studies of various bacterial species,36 gave results that support this interpretation (Figure 3), and the finding is consistent with the fact that these two P. mirabilis strains were both isolated from Greek patients from Athens.



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Figure 3. rep-PCR fingerprinting, generated by REP1R-Dt and REP2-Dt primers, of P. mirabilis isolates. Lanes 1 and 11, molecular size marker (1 kb DNA ladder, Gibco-BRL Life Technologies, Cergy-Pontoise, France); lanes 2–6, P. mirabilis isolates susceptible to ß-lactams used as controls; lane 7, P. mirabilis H223b; lane 8, P. mirabilis 22317; lane 9, P. mirabilis PLAR; lane 10, P. mirabilis 34955.

 
Finally, as reported previously,37,39 while four of the ampC-producing strains studied here were imported, from Greece, Algeria and Egypt, K. pneumoniae 169 is the first clinical strain of this species producing a plasmid-mediated AmpC-type ß-lactamase to be isolated in France from a patient who had not travelled abroad immediately prior to infection.


    Acknowledgements
 
This work was financed by grants from the Ministère de la Recherche (Réseau ß-lactamase, Paris) from the UFR Saint-Antoine, Paris and from the Institut Beecham, La Défense, France.


    Footnotes
 
* Corresponding author. Tel: +33-1-49-28-29-77; E-mail: dominique.decre{at}sat.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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