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Antimicrobial Agents and Chemotherapy, December 2001, p. 3595-3598, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3595-3598.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
-Lactamase from Kluyvera
cryocrescens
Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France
Received 26 February 2001/Returned for modification 23 June 2001/Accepted 28 August 2001
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ABSTRACT |
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A chromosomally located 
-lactamase gene, cloned and expressed in
Escherichia coli from a reference strain of the
enterobacterial species Kluyvera cryocrescens, encoded a
clavulanic acid-inhibited Ambler class A enzyme, KLUC-1, with a pI
value of 7.4. KLUC-1 shared 86% amino acid identity with a subgroup of
plasmid-mediated CTX-M-type extended-spectrum -lactamases (CTX-M-1,
-3, -10, -11, and -12), the most closely related enzymes, and 77%
amino acid identity with KLUA-1 from Kluyvera ascorbata.
The substrate profile of KLUC-1 corresponded to that of CTX-M-type enzymes.
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TEXT |
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At present, the genus
Kluyvera is composed of four enterobacterial species:
Kluyvera ascorbata, Kluyvera cryocrescens,
Kluyvera georgiana, and Kluyvera cochleae
(9). K. ascorbata is more frequently isolated
from clinical specimens, while K. cryocrescens is mostly
isolated from the environment (water, soil, sewage, and hospital
environment) (32). Eighteen detailed cases of human K. cryocrescens infections have been reported, with some
(but not all) of them occurring in immunocompromised patients
(32). The detailed susceptibility of K. cryocrescens to -lactams is not known except for its
resistance to ampicillin (1, 32).
We report here on the characterization of a class A -lactamase from
K. cryocrescens with a substrate profile extended to expanded-spectrum cephalosporins. Sequence analysis revealed its similarity to several plasmid-mediated CTX-M-type extended-spectrum -lactamases (ESBLs).
Bacterial strains and plasmid analysis K. cryocrescens reference strain 79.54 was from the strain collection of the Institut Pasteur (Paris, France). Plasmid DNA extractions, performed as described previously (26), failed to identify plasmids.
Cloning and sequence analysis of -lactamase gene from K.
cryocrescens.
Whole-cell DNA of K. cryocrescens
79.54 was extracted as described previously (26), digested
with Sau3AI, and ligated into the BamHI site of
phagemid pBK-CMV (26). Thirty Escherichia coli
DH10B recombinant clones were obtained after selection on kanamycin-
and amoxicillin-containing plates, as described previously (26). One of the recombinant plasmids that had the
shortest insert (pKC7954) was retained for further analysis. Its DNA
insert (6.1 kb) was sequenced and analyzed as described previously
(26).
-lactamases were identified (Fig.
1) (17). Isoelectric
focusing analysis, performed as reported previously (26),
showed that cultures of K. cryocrescens 79.54 and E. coli DH10B(pKC7954) gave single and identical -lactamases, each
with a pI value of 7.4.
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-lactamase was closely related to the CTX-M-1 (MEN-1)
subgroup of plasmid-mediated enzymes (CTX-M-1, -3, -10, -11, and -12),
sharing 85 to 86% amino acid identity (5, 18, 21). It
shared 77% identity with the CTX-M-2 subgroup (CTX-M-2, -5, -6, and -7 and Toho-1) and 76 and 78% identities with CTX-M-8 and -9, respectively (7, 15, 27, 31).
KLUC-1 from K. cryocrescens shared only 77% amino acid
identity with the chromosomally encoded KLUA-1 -lactamase from
K. ascorbata (GenBank accession no. CAB59824), while the two enterobacterial species from which these -lactamases were
obtained are phylogenetically related (10). The amino acid
identities of KLUC-1 with the naturally occurring class A
-lactamases from Klebsiella oxytoca (3),
Serratia fonticola (22), Citrobacter koseri (formerly Citrobacter diversus)
(24), Proteus vulgaris (23),
Yersinia enterocolitica (29), and
Klebsiella pneumoniae (SHV-1) (4) were 72, 72, 71, 63, 57, and 37%, respectively.
A 1,241-bp ORF was identified 498 bp upstream of
blaKLUC-1 in the same transcription
orientation; this ORF codes for a putative 401-amino-acid protein. This
protein shared 96% identity with an aspartate aminotransferase from
E. coli K-12 (6) and with a putative protein
whose ORF was found upstream of blaKLUA-1
from K. ascorbata (GenBank accession no. 272538).
Additionally, 90% nucleotide identity was found in these intergenic
regions in K. ascorbata and K. cryocrescens. No
upstream-located LysR-type regulator gene was identified, whereas
AmpR genes are upstream-located compared to the chromosomally
encoded -lactamase genes of S. fonticola, P. vulgaris, and C. koseri (14, 16, 20).
Another ORF was identified 928 bp downstream of
blaKLUC-1 in the same transcription
orientation; this ORF encoded a putative protein that shared 69% amino
acid identity with that identified downstream of
blaKLUA-1 (GenBank accession no. 272538).
No consistent nucleotide identity was found in the immediate downstream
region of the -lactamase genes identified in K. cryocrescens and K. ascorbata.
Since the ORFs located upstream and downstream of the -lactamase
genes from K. cryocrescens and K. ascorbata
shared consistent identities, it is possible that they constituted
similar loci. Similarly, AmpC genes in several enterobacterial species
are bracketed by nucleotide sequences that encode functionally related
proteins (25).
A Southern transfer of a gel containing whole-cell DNA of K. cryocrescens 79.54 was performed (28), and the DNA
hybridized by PCR with an 846-bp internal fragment of
blaKLUC-1 as a labeled probe
(28). A positive signal was detected at the chromosomal migration position, further indicating the chromosomal origin of
blaKLUC-1 (data not shown).
Susceptibility testing.
The MICs of selected -lactams were
determined as described previously (26). K. cryocrescens 79.54 was resistant to amoxicillin and ticarcillin
and had reduced susceptibility to cephalothin and cefuroxime (Table
1). It was susceptible to the other
-lactam antibiotics tested. Resistance to aminopenicillins has been
reported previously for K. cryocrescens (1).
Once cloned in pBK-CMV (pKC7954) and expressed in E. coli
DH10B (Table 1), KLUC-1 also conferred resistance or reduced
susceptibility to cefotaxime, ceftriaxone, cefpirome, and aztreonam.
These activities paralleled the activity of a -lactamase from crude
extracts (25) of a culture of E. coli
DH10B(pKC7954), which were 40-fold higher than that of a culture of
K. cryocrescens 79.54 (data not shown). The resistance profile observed for E. coli DH10B(pKC7954)
corresponded to that conferred by plasmid-mediated CTX-M-type
-lactamases, which do not compromise susceptibility to ceftazidime
significantly (31). The addition of clavulanic acid
and tazobactam strongly lowered the -lactam MICs (Table 1). These
results indicate that KLUC-1 is a clavulanic acid-inhibited ESBL that
is likely weakly expressed in K. cryocrescens.
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-lactam resistance pattern conferred by KLUC-1 resembled those
conferred by chromosomally encoded -lactamases of other enterobacterial species: S. fonticola (22),
C. koseri, and P. vulgaris (19).
Biochemical analysis of -lactamase KLUC-1.
A culture of
E. coli DH10B(pKC7954) was grown overnight at 37°C in 2 liters of Trypticase soy broth with amoxicillin (50 µg/ml) and
kanamycin (30 µg/ml). The -lactamase extract was obtained after
sonification as described previously (26). It was further purified by a two-step ultrafiltration procedure, as recommended by the
manufacturer (Vivaspin, 100,000 MWCOPES and 10,000 MWCOPES; Sartorius, Göttingen, Germany). Partially purified
-lactamase was used for kinetic measurements, performed at 30°C in
100 mM sodium phosphate (pH 7.0) as described previously
(25).
-lactamase, measured
with 100 µM cephalothin as the substrate, was 4.2 U · mg of
protein
1 with a 10-fold purification factor.
KLUC-1 had strong activity against benzylpenicillin, piperacillin,
cephalothin, cefuroxime, and ceftriaxone (Table
2). Significant hydrolytic activity was observed against cefotaxime and ceftriaxone, while a very low level of
activity was detectable against ceftazidime. For this substrate, high
Km and low relative
Vmax values were observed (Table 2).
This substrate profile corresponded to that reported for CTX-M-type
enzymes (31).
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-lactamase that may belong to the 2be group of -lactamases of the
Bush-Jacoby-Medeiros classification (8).
Conclusion.
The KLUC-1 -lactamase from K. cryocrescens was mostly related to the subgroup of -lactamases
that comprises CTX-M-1, -3, -10, -11, and -12. However, it was not the
direct progenitor of known plasmid-mediated CTX-M enzymes, in contrast
to KLUA-1 from K. ascorbata, which shares 99% amino acid
identity with the CTX-M-2 -lactamase (C. Humeniuk et al.,
unpublished data [GenBank accession no. CAB59824]). Like the
CTX-M-type enzymes (31), KLUC-1 is a clavulanic
acid-inhibited Ambler class A ESBL that possesses a substrate profile
that includes extended-spectrum cephalosporins but not ceftazidime.
KLUC-1 possesses amino acid residues at key positions that may explain
its extended spectrum of hydrolysis. The serine residue at position 237 may contribute significantly to this extended substrate profile, as
reported for the -lactamase of P. vulgaris
(30). A similar omega loop sequence (residues 161 to 179)
is found for KLUC-1 and the sequences of CTX-M-type enzymes such as
Toho-1 (Fig. 1). The crystal structure analysis of Toho-1 shows that
residue Phe160 suppresses the hydrogen bond between residues Thr160 and
Ser/Thr181 that connects the N and C termini of the omega loop in
non-ESBLs; this may explain, in part, the expanded substrate profile of
Toho-1 (13). Since residue Phe160 was found also in the
KLUC-1 sequence, the lack of a hydrogen bond may also increase the
flexibility of the omega loop, extending the KLUC-1 substrate profile.
Additionally, KLUC-1, like Toho-1 (13), has glycine
residues at positions 232, 236, and 238 that may increase the
flexibility of the B3 strand, which would make it possible for KLUC-1
to bind to bulky extended-spectrum cephalosporins.
-lactamases
in members of the family Enterobacteriaceae (S. fonticola, P. vulgaris, C. koseri, K. ascorbata, and K. cryocrescens) have the same
substrate profile, which includes amino- and ureidopenicillins, cephalothin, cefuroxime, cefotaxime, and ceftriaxone but not
ceftazidime (19). However, KLUC-1 did not confer
resistance to expanded-spectrum cephalosporins in K. cryocrescens. As reported for the expression of the naturally
encoded -lactamase from Klebsiella oxytoca, which is also
independent of a LysR-type regulator (11, 12), it is
likely that extended-spectrum cephalosporin-resistant K. cryocrescens mutants that would contain mutations in the
blaKLUC-1 promoter region may be selected
in vivo. In that case, a strong increase in the level of expression of
the KLUC-1 -lactamase would be expected (as observed when
blaKLUC-1 was cloned on multicopy vector
pBK-CMV [Table 1]), and this would thus confer resistance to
extended-spectrum cephalosporins.
Finally, this report further underlines the fact that enterobacterial
species are natural producers of either no -lactamase or the Ambler
class A and/or class C -lactamase.
Nucleotide sequence accession number. The nucleotide sequence of blaKLUC-1 and the 6.1-kb insert of recombinant plasmid pKC7954 has been assigned GenBank nucleotide database accession no. AY026417.
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ACKNOWLEDGMENTS |
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This work was funded by a grant from the Ministères de l'Education Nationale et de la Recherche (UPRES, grant JE 2227), Université Paris XI, Faculté de Médecine, Paris-Sud, Paris, France.
We thank Chantal Bizet of the Institut Pasteur strain collection for the gift of the K. cryocrescens reference strain and Samuel Bellais for advice in determination of biochemical parameters.
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FOOTNOTES |
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* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr.
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