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Antimicrobial Agents and Chemotherapy, August 2000, p. 2201-2204, Vol. 44, No. 8
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Biochemical-Genetic Characterization and Distribution of OXA-22,
a Chromosomal and Inducible Class D 
-Lactamase from
Ralstonia (Pseudomonas)
pickettii
Patrice
Nordmann,*
Laurent
Poirel,
Maryline
Kubina,
Anne
Casetta, and
Thierry
Naas
Service de Bactériologie-Virologie,
Hôpital de Bicêtre, Assistance Publique/Hôpitaux de
Paris, Faculté de Médecine Paris-Sud, 94275 Le
Kremlin-Bicêtre, France
Received 1 December 1999/Returned for modification 28 March
2000/Accepted 19 May 2000
 |
ABSTRACT |
From genomic DNA of Ralstonia pickettii isolate PIC-1,
a 
-lactamase gene was cloned that encodes the oxacillinase OXA-22. It differs from known oxacillinases, being most closely related to
OXA-9 (38% amino acid identity). The hydrolytic spectrum of OXA-22 is
limited mostly to benzylpenicillin, cloxacillin, and restricted-spectrum cephalosporins. OXA-22-like genes were
identified as single chromosomal copies in five other R. pickettii clinical isolates. The expression of
OXA-22-like -lactamases was inducible in R. pickettii.
| |
TEXT |
Ralstonia pickettii is a
nonfermenting gram-negative rod that is an occasional pathogen of
nosocomial septicemia and tissue infections (10, 17, 20). In
1992, this organism was transferred from the genus
Pseudomonas RNA homology group II to the genus Burkholderia (8) and recently to the novel genus
Ralstonia (22).
The -lactam resistance mechanism(s) of R. pickettii
isolates is not known in detail. In this report, we describe the
genetic and biochemical characterization of inducible oxacillinases
that occur naturally in R. pickettii and that may explain
part of its -lactam resistance profile.
Bacterial strains, PFGE, plasmids, and conjugation assays.
R.
pickettii clinical isolates PIC-1, PIC-2, and PIC-3 were from the
hospitals Bicêtre and Antoine Béclère (Paris area, France), R. pickettii reference strains CIP 103413 and CIP
74.22 were from the strain collection of the Pasteur Institute (Paris, France), and strain ATCC 27511 was from the American Type Culture Collection. These strains were identified by standard biochemical techniques (8).
Comparison of R. pickettii whole-cell DNAs was performed by
a pulsed-field gel electrophoresis (PFGE) technique as previously reported (5, 14). PFGE of either XbaI- or
SpeI-restricted DNAs of R. pickettii strains
showed that they are not clonally related, except for R. pickettii strains PIC-1 and PIC-3 (data not shown).
Plasmid DNA extractions (
3,
13,
14) failed to detect any
plasmid in
R. pickettii strains. Direct transfer (
13,
14)
of an amoxicillin resistance marker from
R. pickettii strains
to rifampin-resistant
E. coli JM109
also
failed.
Susceptibility testing.
MICs of selected -lactams were
determined as described previously (14). R. pickettii isolates were resistant or had decreased susceptibility
to aminopenicillins, ureidopenicillins, restricted-spectrum cephalosporins, ceftazidime, and aztreonam (Table
1), as previously reported
(7). Addition of clavulanic acid and tazobactam did not
significantly modify this resistance profile. Only R. pickettii CIP 103413 was more resistant to piperacillin,
cephalothin, and cefepime (Table 1); tazobactam decreased the
piperacillin MIC 16-fold.
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TABLE 1.
MICs of -lactams for R. pickettii isolates,
E. coli DH10B harboring recombinant plasmid pSC13, and
reference strain E. coli DH10B
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|
Cloning and sequencing of the -lactamase OXA-22 gene.
Genomic DNA of R. pickettii PIC-1 was partially digested
with Sau3AI and ligated into BamHI-digested
phagemid pBK-CMV as previously described (13). Only
one Escherichia coli DH10B strain that contained recombinant
plasmid pSC13 was obtained. Sequence analysis (3) of the
1.2-kb insert of pSC13 revealed an open reading frame (ORF)
of 828 bp (data not shown). The G+C content of this ORF was 65%,
which is within the range of G+C contents of Ralstonia genes
(60.1 to 69.5%; GenBank database).
Within the deduced protein of this ORF (275 amino acids), an S-T-F-K
tetrad was found at class D
-lactamase (DBL) (
6)
positions 71 to 75 (Fig.
1). Four
structural elements characteristic
of DBLs (
2) were found in
this novel enzyme; they were named
OXA-22 (Y-G-N at DBL positions 144 to 146, W-X-E-X-X-L-X-I-S at
DBL positions 164 to 172, Q-X-X-X-L at
positions 176 to 180, and
K-T-G at positions 216 to 218 (Fig.
1)
(
11). In addition, another
stretch of amino acids, at DBL
position 231 to 236, which is conserved
in class D enzymes was also
present (Fig.
1). OXA-22 had low amino
acid identity with other Ambler
DBLs, ranging from 19 to 38% for
OXA-20 to OXA-9, respectively
(
12,
19). The highest percentages
of identity were with
OXA-9, OXA-18, and OXA-12, at 38, 37, and
34%, respectively (
1,
6,
13,
19).
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FIG. 1.
Alignment of the OXA-22 amino acid sequence with those
of the most closely related DBLs and some oxacillinases taken as
representatives of each phylogenetic subgroup of DBLs (6,
10). The shaded boxes indicate regions conserved among DBLs, and
stars indicate highly conserved residues. Dashes indicate gaps in the
alignment.
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|
Biochemical properties of the OXA-22 -lactamase.
Cultures
of E. coli DH10B(pSC13) were grown overnight at 37°C in 6 liters of Trypticase soy broth with amoxicillin at 30 µg/ml and
kanamycin at 30 µg/ml. Unpurified extract of OXA-22 was obtained in
30 ml of sodium phosphate buffer as previously described
(3). This extract was dialyzed in 20 mM
H2SO4-Tris (pH 8) overnight at 4°C and then
loaded onto a preequilibrated Q-Sepharose column (Amersham
Pharmacia Biotech) in Tris
buffer-H2SO4. The -lactamase was eluted with
a linear K2SO4 gradient (0 to 500 mM). The
elution peak containing the highest -lactamase activity was
subsequently dialyzed overnight against 50 mM phosphate buffer, pH 7.0, prior to 10-fold concentration with a Centrisart-C30 microcentrifuge filter (Sartorius, Goettingen, Germany). Kinetic parameters were obtained as described previously (3) using a UV
spectrophotometer with 100 µM cephalothin as the substrate for
inhibition studies, and one unit of enzyme activity was defined
(15) as the activity which hydrolyzed 1.0 µmol of
cephalothin per min.
The specific activity of OXA-22 was 9.1 mU/mg, and its purification
coefficient was 20-fold. OXA-22 showed its strongest hydrolytic
activity against benzylpenicillin, cephalothin, cephaloridine,
and
cloxacillin (Table
2). The lack of
oxacillin hydrolysis by
OXA-22 was peculiar for an oxacillinase
(
4,
11). Further
OXA-22 purification may help to detect
oxacillin hydrolysis as
described for LCR-1 (Y. Yang and K. Bush,
Letter, Antimicrob.
Agents Chemother.
39:1209, 1995). AmpS,
an oxacillinase
related to OXA-12 (90% amino acid identity) from
Aeromonas jandaei,
and LCR-1 strongly hydrolyze oxacillin
but not cloxacillin (
9,
21; Yang and Bush; letter).
Inhibition studies that measured
50% inhibitory concentrations
(IC
50) (
14) showed that OXA-22
activity was
inhibited partially by clavulanic acid (IC
50, 1.2
µM) and
less by tazobactam (IC
50, 6.5 µM). These results
contrasted
with those found for the other oxacillinases, for which the
tazobactam
inhibitory property is equal to or higher than that of
clavulanic
acid (
4,
11). OXA-22 activity was inhibited by
NaCl, as were
other oxacillinases (IC
50, 80 mM)
(
4).
Analytical isoelectric focusing (IEF), performed as previously reported
(
14), revealed that a culture of
R. pickettii
PIC-1
gave two
-lactamase activities with pI values of 7.0 and 7.1,
like
E. coli DH10B(pSC13) cultures (data not shown). These
pI
values may correspond to proteolytic cleavage, partial unfolding,
and/or monomer-dimer conversion of OXA-22. The relative molecular
mass
of OXA-22, determined with an
E. coli DH10B(pSC13)
culture
as previously described (
3), was 28
kDa.
OXA-22-like -lactamases in R. pickettii
isolates.
IEF analysis revealed that the unpurified -lactamase
extract of each R. pickettii culture gave different pI
values: for PIC-3 (as for R. pickettii PIC-1), 7.0 and
7.1; for R. pickettii PIC-2 and ATCC 27511, 7.4; for
R. pickettii CIP 74.22, 7.5. An R. pickettii CIP
103413 culture gave two very different pI values of 6.8 and 7.5, likely
corresponding to two -lactamases.
PFGE of
XbaI-restricted genomic DNAs of
R. pickettii strains and reference strains
Pseudomonas
putida CIP 55.5,
Brevundimonas diminuta CIP 63.27T, and
Comamonas acidovorans 103685 (Pasteur
Institute strain
collection) produced a template used in a Southern
transfer experiment
(
14,
18) with a PCR-obtained 622-bp internal
fragment of
blaOXA-22 as a labeled probe (
3).
blaOXA-22-like
genes were identified in all of
the
R. pickettii isolates but
in none of related
gram-negative species (data not shown). The
hybridizing
XbaI
DNA fragment differed for each
R. pickettii isolate,
except
for
R. pickettii isolates PIC-1 and PIC-3. OXA-22-like
genes
were found at a single copy on a large DNA fragment (>ca.
250 kb),
further underlining the chromosomal origin of these genes
(data not
shown).
Most of the OXA-22-like
-lactamase genes were PCR amplified
(
14) from
R. pickettii genomic DNAs (622 out of
828 bp) as
a result of the choice of internal PCR amplification primers
of
blaOXA-22 (OXA-22A,
5'-TTGCATGAAGGCAAGTGCGACGAG-3'; OXA-22B,
5'-TCAACCTTGTCGTCCTGGATC-3').
The deduced proteins had 96 to
100% amino acid identity with OXA-22.
We identified OXA-22 in
R. pickettii PIC-1 and PIC-3, OXA-22a
in
R. pickettii
PIC-2 and ATCC 27511, and OXA-22b in
R. pickettii CIP 7422 (Table
3). Amplification of an
OXA-22-like gene from
R. pickettii CIP 103413 failed,
despite the use of other primer
combinations and different experimental
setups.
Induction studies.
Induction studies with cefoxitin at 0.5 µg/ml as a -lactam inducer (15) and 100 µM
cephaloridine as the substrate identified induced -lactamase
expression in cultures of all of the R. pickettii isolates
tested. The induction rates ranged from 25- to 64-fold depending on the
strain. In induced cultures, no other -lactamase activity appeared
on an IEF gel, compared to noninduced cultures, thus confirming
that induced -lactamase activities corresponded to those of
OXA-22-like enzymes. Regulation of oxacillinase expression is
known for OXA-12 and AmpS from A. jandaei, which are
chromosomally located and occur naturally (1, 9, 16, 21).
Further work will be directed toward the identification of the
regulatory system of OXA-22-like -lactamases and its comparison with
the two-component regulon identified for coordinated expression of -lactamases of different Ambler classes of A. jandaei
(1, 16).
Nucleotide sequence accession number.
The nucleotide sequence
data reported in this paper will appear in the GenBank nucleotide
database under accession no. AF064820.
| |
ACKNOWLEDGMENTS |
This work was funded by a grant from the Ministère de
l'Education Nationale et de la Recherche (grant UPRES-JE 2227),
Faculté de Médecine Paris-Sud, Université
Paris XI, Paris, France.
| |
FOOTNOTES |
*
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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Antimicrobial Agents and Chemotherapy, August 2000, p. 2201-2204, Vol. 44, No. 8
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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