Service de Bactériologie-Virologie,
Hôpital de Bicêtre, Assistance Publique/Hôpitaux de
Paris, Faculté de Médecine Paris-Sud, 94275 Le
Kremlin-Bicêtre,1 and INSERM
CJF96-06, Faculté de Médecine, 13385 Marseille,2 France
Received 17 August 2000/Returned for modification 12 January
2001/Accepted 3 March 2001
 |
INTRODUCTION |
The most frequent mechanisms of
resistance to extended-spectrum cephalosporins in Pseudomonas
aeruginosa are derepression of the chromosomal AmpC 
-lactamase,
impermeability of the outer membrane, and increased efflux
(5). These various processes, independently or conjointly,
confer resistance to ceftazidime. Extended-spectrum
-lactamases (ESBLs) belonging to each of the Ambler
-lactamase classes are described in P. aeruginosa
(20). Among the clavulanic acid-inhibited Ambler class A
enzymes, TEM- and SHV-related ESBLs, PER-1 and VEB-1 hydrolyze
ceftazidime and cefepime significantly (20). The Ambler
class B enzymes, IMP-1, VIM-1, and VIM-2, have the broadest hydrolysis
profiles, which include hydrolysis of ceftazidime and cefepime
(20, 25). The last group of ESBLs includes OXA-18, the
OXA-2 and OXA-10 derivatives (18, 20), and OXA-24 and
ARI-1, with the last two also hydrolyzing carbapenems (3,
9). Among the OXA-10 variants, OXA-11, -14, and -19 predominantly compromise the activity of ceftazidime, while OXA-17
mainly degrades cefotaxime (8, 18). The OXA-10 variants
confer resistance to cefepime usually at a lower level than they do to
ceftazidime. A recent survey conducted with more than 2,000 isolates of
P. aeruginosa showed that the rates of susceptibility to
ceftazidime and cefepime are similar, being 78.8 to 81.9% and 80 to
83.4%, respectively (27).
The aim of the present work was to characterize the mechanism(s)
involved in a peculiar antibiotic resistance phenotype that combines
resistance to cefepime and susceptibility to ceftazidime.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
P. aeruginosa
SOF-1 was isolated from a rectal swab specimen from a 1-month-old child
hospitalized at the Hôpital de Bicêtre (Le
Kremlin-Bicêtre, France) in 1999. This isolation was the result
of systematic screening for multidrug-resistant gram-negative isolates
from patients admitted to the intensive care units of this hospital. It
was identified with the API 20 NE system (bioMérieux, Marcy
l'Etoile, France). Ciprofloxacin- or rifampin-resistant P. aeruginosa PU21 obtained in vitro and rifampin-resistant
Escherichia coli K-12 C600 obtained in vitro were used as
recipient strains for conjugation, and E. coli DH10B and
P. aeruginosa 104116 (Institut Pasteur Collection, Paris,
France) reference strains (23-25) were used for cloning
experiments. E. coli NCTC 50192 carrying plasmids of 154, 66, 38, and 7 kb served as a control in a plasmid-sizing study
(33). Plasmids pPCRScript-Cam (SK+; Stratagene, Amsterdam, The Netherlands), which carries the chloramphenicol resistance marker,
and the E. coli-P. aeruginosa shuttle vector pBBR1MCS, which
confers resistance to tetracycline, were used for the cloning experiments (12).
Antimicrobial agents and MIC determinations.
Antibiotic
disks (Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) were
used for routine antibiograms. The antimicrobial agents were obtained
from standard laboratory powders and were used immediately after their
solubilization. The agents and their sources were as follows:
amoxicillin, ceftazidime, clavulanic acid, and ticarcillin,
Glaxo-Smith-Kline (Nanterre, France); amikacin, aztreonam, and
cefepime, Bristol-Myers Squibb (Paris-La Défense, France);
cephalothin and moxalactam, Eli Lilly (Saint-Cloud, France); piperacillin and tazobactam, Lederle (Oullins, France); sulbactam, Pfizer (Orsay, France); cefotaxime, cefuroxime, and cefpirome, Aventis
(Paris, France); cefoxitin and imipenem, Merck Sharp & Dohme-Chibret
(Paris, France); and rifampin and chloramphenicol, Sigma (Saint-Quentin
Falavier, France).
MICs were determined by an agar dilution technique on Mueller-Hinton
agar plates with a Steers multiple inoculator and an inoculum of
104 CFU per spot (23). Results of
susceptibility testing were recorded according to the guidelines of the
National Committee for Clinical Laboratory Standards (19)
after incubation at 37°C for 18 h.
Plasmid DNA analysis.
Plasmid DNAs from P. aeruginosa SOF-1 were extracted by two different methods as
described previously (23, 25) and with the Qiagen plasmid
DNA maxi kit (Qiagen, Courtaboeuf, France). Plasmid DNAs were analyzed
by electrophoresis on a 0.7% agarose gel containing 0.5 µg of
ethidium bromide per ml for 16 h at 90 V (28) and
compared to standard sizes of plasmid DNAs of E. coli NCTC
50192. The gel was transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Orsay, France) by the
Southern technique (28). The DNAs were then UV
cross-linked (Stratalinker; Stratagene) for 30 s. The probe, made
of a PCR-generated, 611-bp internal fragment of
blaOXA-31 (see Results section), was labeled
with the an ECL nonradioactive labeling and detection kit (Amersham Pharmacia Biotech), based on a combination of enhanced
chemiluminescence detection and random primer labeling of DNA.
Direct transfer of the ticarcillin and cefepime resistance markers into
rifampin-resistant E. coli K-12 C600 or rifampin- or
ciprofloxacin-resistant P. aeruginosa PU21 was attempted by liquid and solid mating-out assays at 37°C (25). The
antibiotic concentrations for transconjugant selection on Trypticase
soy (TS) agar plates (Sanofi-Diagnostics Pasteur) were 50, 20, 200, and
10 µg/ml for ticarcillin, cefepime, rifampin, and ciprofloxacin, respectively.
Recombinant plasmids were transferred by electroporation into the
E. coli DH10B and P. aeruginosa 104116 reference strains.
Cloning experiments and analysis of recombinant plasmids.
Whole-cell DNA of P. aeruginosa SOF-1 was extracted as
described previously (23). Since most of the oxacillinase
genes are located on a class 1 integron (10, 18), PCR
amplification experiments were attempted with primers located in 5'-CS
and 3'-CS regions (each end of class 1 integrons) (24) and
whole-cell DNA of P. aeruginosa SOF-1 as a template. The
fragment of 3.5 kb obtained by PCR was cloned into the SrfI
site of pPCRScript Cam (SK+) as described previously (25),
and the resulting recombinant plasmid pDAN-1 was selected in E. coli DH10B on Mueller-Hinton agar plates containing amoxicillin
(50 µg/ml) and chloramphenicol (30 µg/ml). Then, the
oxa-31 gene was subcloned by insertion of a
SpeI-PstI fragment of pDAN-1 into the
SpeI-PstI restricted shuttle vector pBBR1MCS,
giving pDAN-2. The oxa-1 gene was amplified by PCR (with
primer OXA1A [5'-AGCCGTTAAAATTAAGCCC-3'] and primer OXA1B
[5'-CTTGATTGAAGGGTTGGGC G-3') with
blaOXA-1-containing plasmid RGN238 from
Salmonella enterica serotype Typhimurium as a template (gift
from R. Labia) (21). The 911-bp fragment encompassing the
oxa-1 gene obtained by PCR was cloned into plasmid pBBR1MCS, giving recombinant plasmid pDAN-3.
DNA sequencing and protein analysis.
Both strands of the
cloned DNA fragments from recombinant plasmids pDAN-1 and pDNA-3 were
sequenced with an Applied Biosystems sequencer (model ABI 373). The
nucleotide and the deduced protein sequences were analyzed with
software available over the Internet at the National Center for
Biotechnology Information website (http://www.ncbi.nlm.nih.gov).
Biochemical analysis.
Cultures of E. coli DH10B
and P. aeruginosa 104116 harboring recombinant plasmids
pDAN-2 and pDAN-3 and cultures of P. aeruginosa SOF-1 were
grown overnight at 37°C in 500 ml of TS broth containing ticarcillin
(50 µg/ml), and -lactamase extracts were obtained as described
previously (24). The -lactamase extracts were suspended
in 20 ml of Tris-100 mM H2SO4-300 mM
K2SO4 buffer (pH 7.0). The protein content was
measured by the Bio-Rad DC Protein assay, and the specific activities
of the -lactamase extracts (except for those from the P. aeruginosa SOF-1 culture) were compared as described previously
(24).
The -lactamase extracts from cultures of E. coli
DH10B(pDAN-2 and pDAN-3) were further purified by a two-step
ultrafiltration as recommended by the manufacturer (Vivapsin, 20 ml;
100,000 MWCOPES and 10,000 MWCOPES; Sartorius, Göttingen,
Germany). Purified -lactamase extracts were used for kinetic
measurements performed at 30°C in Tris-100 mM
H2SO4-300 mM K2SO4
buffer (pH 7.0). The initial rates of hydrolysis were determined with
an ULTROSPEC 2000 UV spectrophotometer (Amersham Pharmacia Biotech), as
described previously (24).
Enzyme preparations from cultures of P. aeruginosa SOF-1 and
the semipurified -lactamases from E. coli DH10B cultures
harboring pDAN-2 and pDAN-3 were also subjected to analytical
isoelectric focusing (IEF) analysis as described previously
(24).
Measurement of norfloxacin and cefepime accumulation.
The
technique used for the efflux study has been described previously
(16, 30). Briefly, exponential-phase P. aeruginosa SOF-1 cells in Luria-Bertani broth (bioMérieux)
were removed by centrifugation and washed once in 50 mM sodium
phosphate buffer (pH 7.0). The pellets were suspended in the same
buffer at a density of 2 × 1010 CFU per ml. Assays
were initiated by adding 14C-norfloxacin (Merck Sharp & Dohme, Rahway, N.J.) or 14C-cefepime (Bristol-Myers Squibb,
Syracuse, N.Y.) at a concentration of 10 µg/ml each (1,010 cpm per
ml). Samples (100 µl each) were removed at set intervals and were
immediately filtered through 0.45-µm-pore-size Whatman GF/C filters
presoaked in phosphate buffer and then washed twice with 5 ml of cold
phosphate buffer. The filters were dried at 80°C, and the
radioactivity was measured in a Beckman scintillation
spectrophotometer. The same experiments were also performed after a
10-min preincubation with the uncoupler carbonyl cyanide
m-chlorophenylhydrazone (CCCP) (Sigma).
Nucleotide sequence accession number.
The nucleotide
sequence of the oxa-31 gene and its class 1 integron has
been assigned EMBL database accession number AF294653.
| |
RESULTS AND DISCUSSION |
Susceptibility testing, plasmid DNA analysis of P. aeruginosa SOF-1, and IEF analysis.
P. aeruginosa
SOF-1 was resistant to cefepime and cefpirome and was susceptible to
ceftazidime, imipenem, and aztreonam (Table 1). Addition of clavulanic acid and
tazobactam did not significantly decrease the MICs of piperacillin and
ticarcillin (Table 1), thus indicating that a Bush group 2b or 2be
-lactamase was not involved (4). Antibiotic
susceptibility testing by disk diffusion showed that P. aeruginosa SOF-1 was also resistant to amikacin, chloramphenicol,
gentamicin, kanamycin, norfloxacin, and tobramycin (data not shown).
The low MIC of ceftazidime was consistent with a weak expression of the
naturally occurring AmpC-type enzyme (5).
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TABLE 1.
MICs of -lactams for P. aeruginosa SOF-1
clinical isolate, P. aeruginosa 104116 harboring recombinant
plasmids pDAN-2 and pDAN-3, reference strain P. aeruginosa
104116, E. coli DH10B harboring recombinant plasmids pDAN-1,
pDAN-2, pDAN-3, and reference strain E. coli DH10B
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Extraction of plasmid DNA from P. aeruginosa SOF-1 gave a
ca. 300-kb plasmid that was not transferred by conjugation to
ciprofloxacin- and rifampin-resistant P. aeruginosa PU21 or
to rifampin-resistant E. coli K-12. It hybridized with an
internal probe for the oxa-31 gene prepared by PCR, thus
showing the plasmid location of this gene (data not shown).
A -lactamase extract of a culture of P. aeruginosa SOF-1
was submitted to IEF analysis and gave two -lactamases with pI values of 7.5 and 8.5, with the latter likely corresponding to an
AmpC-type enzyme (data not shown) (34). A -lactamase
extract of E. coli DH10B(pDAN-1) gave a pI value of 7.5, as
found for P. aeruginosa SOF-1 (data not shown).
Cloning, sequencing of the -lactamase gene, and analysis of the
surrounding sequences.
The cloned fragment of plasmid pDAN-1
encoded three open reading frames (ORFs) located downstream of a class
1 integrase gene (Fig. 1). The first ORF
was 828 bp and encoded a 276-amino-acid preprotein, named OXA-31.
Within the deduced protein of this ORF, a
serine-threonine-phenylalanine-lysine tetrad (S-T-F-K) was found at
positions 70 to 73 according to the class D -lactamase numbering (DBL) (Fig. 2); it was included in the
conserved serine and lysine amino acid residues characteristic of
-lactamases that possess a serine active site or penicillin-binding
proteins (11).
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FIG. 1.
Structure of the class 1 integrons that contain
oxa-31 (A) and oxa-1 (B) cassettes. The arrows
indicate the transcriptional orientations of the ORFs.
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FIG. 2.
Comparison of the amino acid sequence of -lactamase
OXA-31 to those of OXA-1, OXA-4, and OXA-30. Dashes indicate identical
amino acids. The numbering is according to DBL (13). The
highlighted boxes indicate conserved regions within class D
-lactamases.
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Five elements characteristic of class D -lactamases were found:
S-X-V at positions DBL 118 to 120, Y/F-G-X at positions DBL 144 to 146, W-L/I-X-X-X-L-X-I/V at positions DBL 164 to 172, Q-X-X-X-L at positions
176 to 180, and lysine-threonine-arginine (K-T-G) at positions 216 to
218 (Fig. 2) (18). The comparison of the blaOXA-31 nucleotide sequence to those of
blaOXA-1 and blaOXA-30 identified the following nucleotide substitutions: at position 146, T
to C for blaOXA-1 and
blaOXA-30; at position 202, C to G for
blaOXA-1 and blaOXA-30;
at position 391, G to A for blaOXA-1 and
no change for blaOXA-30; and at
position 621, G to T for blaOXA-1 and
blaOXA-30 (21, 31). Compared to
OXA-1 and its derivatives OXA-4 and OXA-30, OXA-31 possessed four, two,
and three amino acid changes, respectively (Fig. 2) (21, 29,
31). None of the amino acid changes that occurred in the OXA-31
sequence when it was compared to the sequences of OXA-1 from S. enterica serotype Tyhimurium, OXA-4 from P. aeruginosa,
and OXA-30 from Shigella flexneri were located at positions
characteristic of class D enzymes (Fig. 2).
Analysis of the surrounding DNA sequences of
blaOXA-31 revealed an ORF located downstream
(Fig. 1). It encoded an aminoacyl adenyl transferase (AADA2a) that
shared 99% amino acid identity with AADA2 that has been associated
with blaPSE-1 from S. enterica serotype Typhimurium (2, 24). This protein conferred
resistance to streptomycin and spectinomycin in E. coli
DH10B (data not shown). An additional ORF located downstream
corresponded to a cmlA-like gene that encodes CMLA6 for
chloramphenicol resistance and that shared 99% amino acid identity
with CMLA1, with only three amino acid changes (data not shown)
(1).
A structure of a class 1 integron was found surrounding the
oxa-31 cassette. The structure consisted of (i) a 5'-CS
containing a class 1 integrase gene with its own promoter,
Pint, (ii) an att11 recombination site, and
(iii) a 3'-CS containing qacE
1 (Fig. 1). The
oxa-31 gene cassette had a core site (5'-GTTGGGC-3'), an inverse core site (5'-GCCCAAC-3'), and a 59-base
element made up of 108 bp of the sequence downstream of this gene that
was identical to that found in the oxa-1 cassette
(21). The 182 N-terminal amino acids of the integrase
identified from the sequenced part of the corresponding ORF were
identical to those found in several class 1 integrons (6,
32). Compared to the corresponding promoter region for
blaOXA-1, the P1 promoter sequence
of blaOXA-31 differed by one substitution in the
10 sequence, and the P2 promoter sequence of the
blaOXA-31 integron was probably not functional due to a shorter spacing (14 bp instead of 17 bp). This promoter pair
was identical to the hybrid promoter sequences found in the blaOXA-2-containing integron (7, 14,
21).
Comparison of OXA-31 and OXA-1 activities.
The MIC of cefepime
for E. coli DH10B(pDAN-1) increased slightly compared to
that for E. coli DH10B, while the MICs of ceftazidime remained unchanged and the MICs of cefotaxime were only weakly increased (Table 1). To show that OXA-31 confers resistance to cefepime
and susceptibility to ceftazidime, a series of experiments was
performed. Recombinant plasmid pDAN-2 that contained a
blaOXA-31-containing DNA fragment from pDAN-1
was transferred by electroporation into reference strains E. coli DH10B and P. aeruginosa 104116. The MICs of
cefepime, cefpirome, and ticarcillin were significantly increased for
P. aeruginosa 104116(pDAN-2) compared to those for P. aeruginosa 104116, while the MICs of ceftazidime and cefotaxime remained unchanged or were slightly increased, respectively (Table 1).
This experiment clearly established the role of OXA-31 in the
acquisition of resistance to cefepime in P. aeruginosa.
Recombinant plasmid pDAN-3, which had a
blaOXA-1-containing DNA fragment, was
transferred by electroporation into reference strains E. coli DH10B and P. aeruginosa 104116. The MICs of
-lactams for E. coli DH10B harboring pDAN-3 (OXA-1) and
pDAN-2 (OXA-31) on the one hand and for P. aeruginosa 104116 harboring pDAN-2 and pDAN-3 on the other were identical (Table 1). The
specific activities of -lactamase extracts for several -lactams
were determined with extracts from E. coli DH10B and
P. aeruginosa 104116 cultures expressing either OXA-31 or
OXA-1 (Table 2). The values obtained were
almost identical, showing that OXA-1 and OXA-31 had similar hydrolysis
spectra for cefepime and cefpirome on the one hand and for ceftazidime
and cefotaxime on the other, although for the last two drugs hydrolysis
was weak or undetectable (Table 2).
The specific activities obtained from P. aeruginosa cultures
were much higher compared to those obtained from E. coli
cultures, whatever substrate was used, except for ceftazidime and
aztreonam. Increases ranged from 10- to 15-fold according to the
substrate, probably as a result of a higher rate of plasmid replication
in P. aeruginosa cultures or better -lactamase folding.
The results for the cefepime and cefpirome specific activities
correlated to the MICs, with a marked increase in the level of
resistance in P. aeruginosa (Tables 1 and 2).
The hydrolysis parameters for OXA-1 and OXA-31 showed that they are
typical oxacillinases (Table 3). Kinetic
parameters showed that the catalytic activities of OXA-1 and OXA-31
were similar and were significant toward cefepime (Table 3).
Efflux study.
The efflux of cefepime has been reported in
P. aeruginosa as a resistance mechanism that may be specific
to cefepime and cefpirome (26). Thus, the efflux of
cefepime was examined as an additional mechanism of resistance to
cefepime in P. aeruginosa SOF-1. The levels of norfloxacin
and cefepime that accumulated at steady state were compared in the
absence or in the presence of the uncoupler CCCP since intracellular
uptake is energy independent and efflux is energy dependent. The
steady-state level of intracellular norfloxacin increased by about
threefold in the presence of CCCP, thus indicating the presence of an
efflux mechanism for this drug that paralleled the resistance to
norfloxacin observed in P. aeruginosa SOF-1 (Fig.
3A). By contrast, the addition of CCCP
did not change the intracellular concentration of cefepime
significantly, thus ruling out efflux of this drug in P. aeruginosa SOF-1 (Fig. 3B).
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FIG. 3.
Intracellular accumulation of cefepime and norfloxacin
in P. aeruginosa clinical isolate SOF-1. The intracellular
concentrations of radiolabeled norfloxacin (A) and cefepime (B) were
measured in cells of P. aeruginosa SOF-1 with ( ) or
without ( ) the energy uncoupler CCCP. Values are means of two
independent experiments.
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Conclusion.
We showed that OXA-31 confers resistance to
cefepime and susceptibility to ceftazidime in P. aeruginosa
SOF-1, a property not reported previously (13). This
property is shared by OXA-1 as well and is very likely shared by other
OXA-1 derivatives such as OXA-4 (17). Indeed,
-lactamase OXA-4, originally described in P. aeruginosa
(15, 22) and reported recently from several P. aeruginosa isolates in Japan, confers resistance to the oxyimino cephalosporin cefclidin and susceptibility to ceftazidime
(17). Thus, OXA-1 and its derivatives may selectively
hydrolyze some 2-amino-5-thiazolyl cephalosporins (cefpirome, cefepime,
and cefclidin) and not others (ceftazidime, cefotaxime), with these
drugs mostly differing by substitutions at the C-3 of the cephem core.
Finally, this study showed that in a routine laboratory ceftazidime
resistance should not be reported on the sole basis of cefepime
resistance for P. aeruginosa-producing OXA-1 derivatives, as
opposed to those that produce class A ESBLs. This result has clinical
implications for the choice of the most appropriate treatment for
nosocomial infections due to P. aeruginosa.
This work was funded by a grant from the Ministère de
l'Education Nationale et de la Recherche (UPRES, grant JE-2227),
Université Paris XI, Paris, France; INSERM (grant CJF 96-06),
Marseilles, France; and a grant-in-aid from Glaxo-Smith-Kline (from F. Leblanc), Paris, France.
We thank M. Malléa for help with the efflux experiments and A. Ladzunski for gift of plasmid pBBR1MCS.
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Abstract
Introduction
