Novel, plasmid-encoded, TEM-derived extended-spectrum beta-lactamase in Klebsiella pneumoniae conferring higher resistance to aztreonam than to extended-spectrum cephalosporins

Abstract

A clinical isolate of Klebsiella pneumoniae was more resistant to aztreonam than to cefotaxime and ceftazidime. It produced
a clavulanate-susceptible beta-lactamase with an isoelectric point of 6.3 which readily hydrolyzed penicillins, cefotaxime,
and ceftazidime, but which hydrolyzed aztreonam poorly. The enzyme was encoded by a gene on a 15-kb plasmid; the gene hybridized
with an intragenic DNA probe of blaTEM.

Full text

ANTIMICROBIAL
AGENTS
AND
CHEMOTHERAPY,
Sept.
1993,
p.
2020-2023
0066-4804/93/092020-04$02.00/0
Copyright
©
1993,
American
Society
for
Microbiology
Novel,
Plasmid-Encoded,
TEM-Derived
Extended-Spectrum
,B-Lactamase
in
Klebsiella
pneumoniae
Conferring
Higher
Resistance
to
Aztreonam
than
to
Extended-Spectrum
Cephalosporins
GUILLAUME
ARLET,1*
MARTINE
ROUVEAU,1
GENEVIEVE
FOURNIER,2
PHILIPPE
H.
LAGRANGE,1
AND
ALAIN
PHILIPPON'
Service
de
Microbiologie,
H6pital
Saint-Louis,
75475
Paris
Cedex
10,
1
and
Laboratoire
de
Bacteriologie,
Faculte
de
Me6decine
Cochin-Port-Royal,
75674
Paris
Cedex
14,2
France
Received
4
Februaxy
1993/Returned
for
modification
19
April
1993/Accepted
6
July
1993
A
clinical
isolate
of
Kiebsiella
pneumoniae
was
more
resistant
to
aztreonam
than
to
cefotaxime
and
ceftazidime.
It
produced
a
clavulanate-susceptible
13-lactamase
with
an
isoelectric
point
of
6.3
which
readily
hydrolyzed
penicillins,
cefotaxime,
and
ceftazidime,
but
which
hydrolyzed
aztreonam
poorly.
The
enzyme
was
encoded
by
a
gene
on
a
15-kb
plasmid;
the
gene
hybridized
with
an
intragenic
DNA
probe
of
blaTEM.
The
development
of
highly
stable
extended-spectrum
cephalosporins
at
the
beginning
of
the
1980s
was
a
major
therapeutic
advance.
Some
years
later,
transferable
extend-
ed-spectrum
,-lactamases
were
identified
mainly
in
nosoco-
mial
isolates
of
Kiebsiella
pneumoniae
(for
reviews,
see
references
5
and
10).
Such
enzymes
have
been
shown
to
be
derived
from
SHV-
or
TEM-type
,B-lactamases
by
one
or
more
amino
acid
substitutions
(10,
13).
These
extended-
spectrum
P-lactamases
confer
three
resistance
phenotypes
(CTX,
CAZa,
and
CAZb)
according
to
the
level
of
resistance
to
cefotaxime,
ceftazidime,
and
aztreonam
(2).
We
describe
here
a
novel
extended-spectrum
P-lactamase
produced
by
a
clinical
isolate
(1989)
of
K
pneumoniae
(SLK52)
recovered
in
our
hospital
from
a
patient
with
a
urinary
tract
infection.
The
isolate
was
found
to
be
more
resistant
to
aztreonam
than
to
cefotaxime
or
ceftazidime
(ATM
phenotype).
Conjugation
experiments
were
performed
by
mixing
equal
volumes
(1
ml)
of
exponentially
growing
cultures
of
SLK52
and
Escherichia
coli
K-12
J53-2
resistant
to
rifampin
as
described
elsewhere
(6).
A
second
conjugation
experiment
between
transconjugants
E.
coli
K-12
J53-2
and
E.
coli
K-12
C600
recipient
(resistant
to
nalidixic
acid)
was
done
under
the
same
conditions,
and
a
third
conjugation
experiment
was
done
between
transconjugants
E.
coli
C600
and
E.
coli
K-12
HB101
(resistant
to
streptomycin).
E.
coli
K-12
HB101
was
used
as
the
recipient
strain
for
transformation
experiments.
Competent
cells
were
prepared
by
treatment
with
calcium
chloride
(8).
The
resistance
to
aztreonam,
oxyimino-ceph-
alosporins,
and
penicillins
was
transferred
by
conjugation,
first
from
K
pneumoniae
SLK52
to
E.
coli
J53-2
with
a
high
frequency
(10-3)
and
then
from
E.
coli
J53-2
to
E.
coli
C600,
but
not
from
E.
coli
C600
to
E.
coli
HB101.
It
was
trans-
ferred
from
E.
coli
C600
to
E.
coli
HB101
only
by
transfor-
mation.
No
resistance
determinants
other
than
P-lactam
resistance
were
transferred
in
any
experiment.
P-Lactamase
was
assayed
in
sonicated
extracts
of
the
resistant
K
pneumoniae
strain
and
E.
coli
C600
transcon-
jugants.
Isoelectric
focusing
was
performed
in
polyacryl-
amide
gels
as
described
previously
(9).
1-Lactamase
activity
was
subsequently
located
on
the
same
gel
by
the
classic
*
Corresponding
author.
nitrocefin
method
and
the
iodine-starch
agar
procedure
(3,
11).
The
pI
of
the
enzyme
was
determined
by
comparison
with
the
pIs
of
reference
1-lactamases:
TEM-1
(Rill),
CTX-1-TEM-3
(pCFF04),
and
SHV-1
(p453).
The
pl
was
estimated
to
be
6.3
(Fig.
1).
P-Lactam
hydrolytic
activity
was
evaluated
by
a
microa-
cidimetric
method
(6).
The
substrate
profile
of
the
enzyme
was
determined
by
using
13
P-lactams,
with
the
Vm.
values
being
expressed
relative
to
that
of
benzylpenicillin.
The
substrate
profile
of
this
novel
,-lactamase
compared
with
that
of
TEM-3
(pl,
also
6.3)
is
reported
in
Table
1.
Both
the
novel
P-lactamase
and
TEM-3
hydrolyzed
amino-
and
car-
boxypenicillins.
In
contrast,
the
novel
,-lactamase
showed
lower
activities
against
cefotaxime,
ceftriaxone,
and
ceftazi-
dime
than
did
the
TEM-3
P-lactamase.
No
activity
against
aztreonam
(compared
with
the
hydrolysis
observed
with
the
,B-lactamase
of
K.
oxytoca
SL7811
resistant
to
aztreonam,
9%),
cefoxitin,
moxalactam,
or
imipenem
was
detected.
Because
the
hydrolysis
of
aztreonam
associated
with
high
MIC
(Table
2)
was
not
detected
by
the
microacidimetric
method,
P-lactamase
activity
was
determined
spectrophoto-
metrically
at
30°C
in
10
mM
phosphate
buffer
(pH
7.0)
by
using
a
double-beam
spectrophotometer
(model
550S;
The
Perkin-Elmer
Corp.).
The
following
wavelengths
were
used
in
the
present
study:
for
cephaloridine,
260
nm;
for
aztreo-
nam,
318
nm.
The
rate
of
hydrolysis
of
aztreonam
relative
to
that
of
cephaloridine
was
ninefold
greater
for
the
novel
enzyme
(1.25%)
compared
with
that
for
TEM-3
(0.14%).
Inhibition
of
P-lactamase
activity
by
cloxacillin
(0.1
mM),
clavulanic
acid
(0.01
,M),
sulbactam
(1
,uM),
and
chloride
ions
(100
mM)
was
expressed
as
the
percentage
of
inhibition
of
the
rate
of
benzylpenicillin
hydrolysis.
The
inhibitory
activities
of
clavulanic
acid
and
sulbactam
were
determined
after
preincubation
with
the
enzyme
for
10
min
at
37°C.
Activity
was
strongly
inhibited
by
clavulanic acid
(43%)
and
was
partially
inhibited
by
sulbactam
(46%)
and
cloxacillin
(60%).
Chloride
ions
were
not
good
inhibitors.
Moreover,
the
concentration
of
aztreonam
required
to
inhibit
50%
of
the
benzylpenicillin
hydrolysis
(IC50)
was
determined
without
preincubation
for
TEM-2,
TEM-3,
and
the
novel
P-lacta-
mase
by
the
microacidimetric
method.
The
IC50
determina-
tions
(Table
1)
showed
that
the
novel
P-lactamase
was
8.5
2020
Vol.
37,
No.
9

NOTES
2021
7.7-
6.3
5~~~~~~~~~~
.4
A
*
C
D
E
FIG.
1.
Comparative
analytical
isoelectric
focusing
patterns
of
the
novel
1-lactamase
from
the
K
pneumoniae
donor
SLK52,
E.
coli
C600
transconjugant,
E.
coli
HB101
transformant,
and
reference
P-lactamases.
Lanes:
A
and
E,
TEM-1
(R1ll;
pl,
5.4),
TEM-3
(pCFF04;
pI,
6.3),
and
SHY-1
(p453,
pl,
7.7);
B,
K
pneumoniae
SLK52;
C,
E.
coli
C600
transconjugant;
D,
TEM-4
(pUD16;
pI,
5.9)
and
SHV-5
(pAFF2;
pl,
8.2);
and
F,
E.
coli
HB101
transformant.
and
15.5
times
more
susceptible
to
inhibition
by
aztreonam,
than
were
TEM-3
and
TEM-2,
respectively.
The
MICs
of
the
,-lactams
were
determined
in
the
pres-
ence
or
absence
of
clavulanic
acid
by
an
agar
dilution
method
by
using
a
multi-inoculator
device
(MIC
2000;
Dynatech
Laboratories,
Inc.)
and
Mueller-Hinton
agar.
Plates
inoculated
with
105
to
106
CFU
were
incubated
at
37°C
for
18
h.
The
MICs
of
,-lactam
antibiotics
are
reported
in
Table
2.
The
MICs
of
aztreonam
for
the
clinical
K
pneumoniae
isolate
and
the
E.
coli
K-12
transconjugants
and
transformants
were
8-
to
32-fold
greater
than
those
of
cefotaxime
and
ceftazidime.
This
is
in
contrast
to
the
pat-
terns
of
resistance
observed
for
TEM-3,
CAZ-7,
TEM-14,
TEM-16,
and
TEM-18
transconjugants
harboring
P-lacta-
mases
with
pIs
of
6.3
conferring
the
CTX
or
CAZ
pheno-
types
(4,
7,
12).
Significant
potentiation
of
activity
was
observed
with
clavulanic
acid
for
K
pneumoniae
SLK52
(MICs
of
aztreonam,
cefotaxime,
and
ceftazidime
were
reduced
by
512-,
32-,
and
64-fold,
respectively)
and
the
E.
coli
K-12
recipients.
Strain
SLK52
was
susceptible
to
mox-
alactam
and
imipenem
and
was
moderately
susceptible
to
cefoxitin.
Plasmid
DNA
was
extracted
by
an
alkaline
lysis
procedure
(15),
and
electrophoresis
was
performed
on
0.7%
agarose
gels
(Fig.
2).
Only
one
plasmid,
pSLH52,
was
detected
in
all
four
strains,
although
larger
plasmids
were
present
in
the
K
pneumoniae
clinical
isolate
and
the
E.
coli
J53-2
transcon-
jugant.
For
restriction
endonuclease
analysis,
DNA
was
purified
on
a
cesium
chloride-ethidium
bromide
density
gradient
(8).
The
restriction
endonucleases
EcoRI
and
HindIII
were
purchased
from
Bethesda
Research
Laboratories
(Paris,
France).
Enzymatic
reaction
conditions
were
those
recom-
mended
by
the
manufacturer.
The
generated
fragments
were
separated
by
electrophoresis.
The
1-kb
ladder
from
Be-
thesda
Research
Laboratories
and
X
phage
digested
with
HindIII
were
used
as
molecular
size
standards
for
estima-
tions
of
the
sizes
of
the
linear
fragments.
The
estimated
size
of
pSLH52
was
15
kb
(data
not
shown).
A
TEM-specific
DNA
probe
was
obtained
by
amplification
of
an
internal
504-nucleotide
fragment
of
the
bla
gene
of
pBR322
and
purification
after
electrophoresis
in
2%
low-
melting-point
agarose
as
described
previously
(1).
The
DNA
probe
was
labeled
with
[32P]dCTP
by
using
the
Amersham
multiprime
system.
Hybridization
was
carried
out
under
stringent
conditions
after
the
transfer
of
plasmid
DNA
to
a
nylon
membrane
by
the
method
of
Southern
(14).
RP4
(TEM-2)
and
pIP173
(SHV-1)
were
used
as
controls.
Hybrid-
ization
analysis
confirmed
that
the
P-lactamase
gene
was
located
on
pSLH52
and
that
it
cross-hybridized
with
the
probe
derived
from
the
TEM-type
family
(Fig.
2).
Among
the
extended-spectrum
1-lactamases
that
have
been
described
(5,
10),
the
following
three
resistance
pheno-
types
could
be
characterized:
CTX,
CAZa,
and
CAZb
(2).
The
susceptibility
pattern
determined
by
this
novel
enzyme
clearly
distinguishes
a
phenotype
not
previously
described
(ATM
resistance
phenotype).
Low
MICs
of
cefoxitin,
mox-
alactam,
and
imipenem
and
a
high
potentiation
effect
of
clavulanate
on
the
MICs,
when
associated
with
oxyimino-1-
lactams,
have
been
also
reported
for
other
extended-spec-
trum
,B-lactamases.
A
pl
of
6.3
does
not
allow
the
differen-
tiation
of
this
enzyme
from
some
extended-spectrum
P-lactamases
(TEM-3,
TEM-14,
TEM-16,
TEM-18,
and
CAZ-7)
(4,
7,
12).
However,
its
substrate
profile
is
charac-
teristic:
its
rates
of
hydrolysis
of
penicillins
are
similar
to
those
of
TEM-
and
SHV-type
enzymes;
however,
its
activity
against
oxyimino-cephalosporins
is
different
from
that
of
TEM-3,
particularly
for
cefotaxime
and
ceftazidime;
a
low
TABLE
1.
Comparative
enzymatic
properties
of
the
novel
extended-spectrum
P-lactamase
and
TEM-3
I-Lactamasea
Property
TEM-3
Novel
pI
6.3
6.3
Enzymatic
activity
against":
Amoxicillin
47
97
Carbenicillin
16
16
Mezlocillin
260
232
Oxacillin
5
2
Cloxacillin
1
1
Cephaloridine
295
411
Cefotaxime
595
130
Ceftriaxone
216
52
Ceftazidime
34
10
Moxalactam
<1
<0.5
Cefoxitin
<1
<0.5
Aztreonam
<1
<0.05
Imipenem
<1
<0.5
Inhibition
(%)
byc:
Cloxacillin,
0.1
mM
66
60
Clavulanate,
1
FLM
92
100
Clavulanate,
0.01
,uM
43
Sulbactam,
1
pM
63
46
NaCl,
100
mM
11
0
IC50
by
aztreonam
(P.M)d
325
38
a
Sonicated
extracts
of
E.
coli
K-12
(transconjugants).
bVmax
relative
to
that
of
benzylpenicillin,
which
was
set
as
100%.
c
Percent
inhibition
with
benzylpenicillin
as
the
substrate.
d
Concentration
required
to
inhibit
the
hydrolysis
of
benzylpenicillin
by
50%
(TEM-2,
580
pM).
VOL.
37,
1993

ANTIMICROB.
AGENTS
CHEMOTHER.
TABLE
2.
MICs
of
P-lactam
antibiotics
for
strains
harboring
different
extended-spectrum
P-lactamases
(pI
6.3)
in
the
presence
or
absence
of
clavulanic
acida
MIC
(Lg/ml)
Strain
Plasmid-mediated
AMX
CTX
CAZ
ATM
13-lactamase
______
_____
CFT
FOX
MOX
IMP
_-
+
-
+
+
+
K
pneumoniae
SLK52
Novel
>8,192
32
8
0.25
32
0.5
256
0.5
128
16
1
0.12
E.
coli
C600
8
8
0.06
0.06
0.12
0.12
0.06
0.06
4
4
0.12
0.12
E.
coli
C600
Novel
8,192
8
4
0.06
8
0.25
128
0.25
32
4
0.25
0.12
E.
coli
C600
TEM-3
8,192
8
8 0.06
16
0.5
8
0.12
128
4
0.25
0.12
E.
coli
C600
CAZ-7
8,192
8
1
0.06
128
0.5
16 0.12
16
4
0.25
0.12
E.
coli
C600
TEM-14
8,192
8
4
0.12
8
0.5
4
0.12
128
4
0.25
0.12
E.
coli
C600
TEM-16
4,096
8
2
0.06
4
0.25
2
0.06
32
4
0.12
0.12
E.
coli
C600
TEM-18
4,096
8
2
0.06
4
0.25
2
0.06
32
4
0.12
0.12
E.
coli
HB101
8
4
0.03
0.03
0.06 0.06
0.03
0.03
2
1
0.06
0.06
E.
coli
HB101
Novel
8,192
8
4
0.03
4
0.25
128
0.06
32
1
0.12
0.12
a
AMX,
amoxicillin;
CTX,
cefotaxime;
CAZ,
ceftazidime;
ATM,
aztreonam;
CFT,
cephalothin;
FOX,
cefoxitin;
MOX,
moxalactam;
IPM,
imipenem;
-,
absence
of
clavulanic
acid
(2
±g/ml);
+,
presence
of
clavulanic
acid
(2
,ug/ml).
rate
of
hydrolysis
of
aztreonam
was
detected
(only
by
spectrophotometric
determination),
as
reported
previously
for
many
of
the
extended-spectrum
1-lactamases
(10).
The
comparative
inhibition
profile
cannot
differentiate
this
en-
zyme
from
other
extended-spectrum
f-lactamases,
but
IC50s
suggest
that
this
novel
enzyme
has
a
strong
affinity
for
aztreonam
when
compared
with
that
of
TEM-2
or
TEM-3.
The
genetic
determinant
of
this
new
enzyme
initially
seemed
to
be
transferable
and
carried
by
a
small
plasmid
of
15
kb.
In
fact,
this
plasmid
was
not
conjugative
and
did
not
cotransfer
resistance
to
the
other
drugs
tested;
this
is
unusual,
because
most
extended-spectrum
P-lactamases
are
encoded
by
a
self-transferable
plasmid
(5)
and
may
explain
the
absence
of
spread
of
this
enzyme
to
other
strains
in
our
hospital.
The
novel
extended-spectrum
,-lactamase
described
here
seems
to
derive
from
TEM-type
enzymes
and
to
be
respon-
sible
for
a
new
resistance
phenotype,
suggesting
another
possibility
of
evolution
of
such
enzymes.
We
propose
that
this
enzyme
be
called
TEM-22.
Determination
of
the
nucle-
otide
sequence
of
its
gene
will
be
required
to
identify
the
1
2
3
4
5
6
A
1
2
3
4
5
6
v
_01
B
FIG.
2.
(A)
Agarose
gel
electrophoresis
of
crude
DN4A
lysates
from
the
K
pneumoniae
donor
SLK52,
E.
coli
J53-2
and
C600
transconjugants,
and
E.
coli
HB101
transformant.
Lanes:
1,
pIP173
(SHV-1);
2,
RP4
(TEM-2);
3,
E.
coli
HB101;
4,
E.
coli
C600;
5,
E.
coli
J53-2;
and
6,
K
pneumoniae
SLK52.
(B)
The
corresponding
autoradiograph
after
transfer
onto
a
nylon
filter
and
hybridization
with
the
TEM
probe.
precise
nature
of
the
amino
acid
substitution(s)
in
this
enzyme
and
to
establish
it
as
unique.
We
are
grateful
to
D.
Sirot,
C.
Mabilat,
and
P.
Courvalin
for
supplying
strains
and
to
L.
Gutmann
and
E.
Collatz
for
the
spec-
trophotometric
determinations.
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NOTES

NOTES
2023
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G.
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and
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to
third-generation
cephalo-
sporins
due
to
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VOL.
37,
1993