Journal of Antimicrobial Chemotherapy (1996) 38, 183-191

Characterization of an inhibitor-resistant enzyme IRT-2 derived from

TEM-2 /Mactamase produced by Proteus mirabilis strains
L. Brer", C. Chanal', D. Sirof, R. Labia' and J. Sirof

Ten clinical isolates of Proteus mirabilis were found to produce an inhibitor-resistant

TEM /J-lactamase (IRT) in association with a TEM-1 enzyme. The IRT enzyme was
derived from TEM-2. The blaua gene differs from blareMi gene by one point mutation
which leads to the amino-acid substitution Arg->Ser at position 244, as observed for
the original IRT-2 enzyme derived from TEM-1 reported in Escherichia coti. This
is the first report of an IRT /?-lactamase derived from TEM-2.

Introduction

Since 1989, the TEM-derived /?-lactamases that exhibit reduced activities against
/?-lactam antibiotics and resistance to /Mactamase inhibitors (IRT enzymes) have been
observed mainly in Escherichia coli strains (Blasquez et al., 1993; Belaaouaj et al., 1994;
Bran et al., 1994; Sirot et al., 1994; Zhou et al., 1994; Henquell et al., 1995). More
recently, IRT enzymes have been described in two Klebsiella pneumoniae strains
(Lemozy et al., 1995). All IRT enzymes previously reported were TEM-1 derivatives.
This report describes an IRT enzyme detected in ten clinical isolates of Proteus mirabilis.
Material and methods

Bacterial strains
Ten P. mirabilis strains were isolated during the first six months of 1995 from different
patients in different wards. P. mirabilis CF 1709, the first isolate, was cultured in
abdominal pus from a patient hospitalized in a gastroenterology unit in the teaching
hospital of Clermont-Ferrand, France.
Susceptible P. mirabilis strains (ATCC 103181T) and TEM-1 (CF 019) or TEM-2 (CF
029) producers were used for comparison of inhibition zones and MIC values.
Susceptibility testing and resistance phenotype
Susceptibility testing was performed by the disc diffusion method according to the
recommendations of the Antibiogram Committee of the French Society for
0305-7453/96/080183 + 09 $12.00/0

183
1996 The British Society for Antimicrobial Chemotherapy

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'Laboratoire de Bacteriologie, Faculte de Medecine, Universite cTAuvergne, 28 Place

Henri-Dunant, 63001 Clermont-Ferrand; bMuseum National cTHisloire Naturelle
CNRS URA 401, 63 rue Buffon, 75231 Paris Cedex 05, France

184

L. Bret et at.

Microbiology (Acar et al., 1995) and by the rapid ATB-E method (Biomerieux, Marcy
l'Etoile, France) for IRT detection (Bret et al., 1994).
The MICs of amoxycillin, amoxycillin-clavulanate, ticarcillin, ticarcillin-clavulanate,
piperacillin, piperacillin-tazobactam (clavulanic and tazobactam at fixed concentrations
of 2 and 4 mg/L respectively) and cephalothin were determined by a dilution method
on Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and
an inoculum of 10*cfu per spot. Antibiotics were provided as powders by SmithKline
Beecham Pharmaceuticals (Worthing, UK), Lederle Laboratories, (Groupe Cyanamid,
France).
Isoelectric focusing

Transfer experiment
A transfer experiment was performed for the strain CF 1709 with recipient strain
ATCC 10318IT. The transconjugant was selected on agar containing nalidixic acid
(150 mg/L) and gentamicin (8 mg/L) or amoxycillin-clavulanate (64 mg/L).
Sequencing of DNA amplified by PCR
On the assumption that the strain contained blaJEM, single-stranded DNA template was
generated for sequencing by PCR, performed with an asymmetric ratio of the
amplification primers A and B. The nucleotide sequence was determined as previously
described (Chanal et al., 1992) by direct sequencing of the amplified product obtained
from P. mirabilis strain CF 1709 and its P. mirabilis transconjugant.
Determination of /Mactamase kinetic constants (A"m, V^*)
The Km and V^ constants of the /Mactamases were obtained by computerized
micro-acidimetric method described by Labia, Andrillon & Le Goffic (1973). The A"m
and Vmn values of the plasmid-mediated /Mactamases were determined by using
partially purified extracts obtained by culture of P. mirabilis transconjugant and
compared with those of the /Mactamases TEM-1, TEM-2 and IRT-2. The relative Vmtl
rate of hydrolysis for amoxycillin, ticarcillin and cephalothin were compared with that
for benzylpenicillin, which was taken as 100%.
Ribotyping
Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA)
region analysis were obtained for the ten isolates according the following procedure.
For the isolation of DNA, isolates were grown in 20 mL of Mueller-Hinton broth
overnight at 37C, and centrifuged. After extraction with guanidine hypochloride and
ammonium acetate, the DNA was precipitated with absolute ethanol, chilled at 20C,
and redissolved in Tris HCl-EDTA-NaCl solution.
The procedure used for RFLP of rDNA region analysis was that of Bingen et al.

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Isoelectric focusing was performed with polyacrylamide gels containing ampholines

with a pH range 3.5-10.0, as previously described (Sirot et al., 1994). /Mactamases of
known pi TEM-1 (5.4), TEM-2 (5.6), IRT-2 (5.2) were used as standards.

Inhibitor-resistant TEM in P. mirabilis

185

(1993). Strains P. mirabilis ATCC 103181T and P. mirabilis TEM-1 (strain CF 019)
were used for comparison.

Results and discussion

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The resistance phenotype (MICs) of P. mirabilis CF 1709 and of its transconjugant

was characterized by high level resistance to amoxycillin (512-256 mg/L) and
amoxycillin-clavulanate (256-128 mg/L) and by low level resistance to ticarcillin
(32-4 mg/L) (Table I). These strains were susceptible to ticarcillin-clavulanate
(4-2 mg/L), piperacillin (4-2 mg/L) and cephalothin (8-2 mg/L). The phenotype
differed from that of TEM-1 and TEM-2 P. mirabilis producers (CF 019 and CF 029).
The latter is characterized by lower MICs of amoxycillin-clavulanate (4-32 mg/L) and
higher MICs of ticarcillin (128-1024 mg/L).
In isoelectric focusing of P. mirabilis CF 1709 and of its transconjugant only one band
of pi 5.4 was observed. Nucleotide sequencing revealed the presence of two different
WOTEM genes in the clinical isolate CF 1709 which might encode for a TEM-1 enzyme
of pi 5.4 and a mutated TEM-2 enzyme (IRT) of the same pi. In the transconjugant,
nucleotide sequencing revealed only the Wa,RT gene. This gene was identical to the
blaTtM-2 gene at positions 226, 317, 346, 436, 604, 682 and 925 which discriminate the
bla-rEM genes (Chen & Clowes, 1987; Goussard & Courvalin, 1991). Only one mutation,
the nucleotide change C->A at position 317, results in an amino-acid substitution Gin
(TEM-l)->Lys (TEM-2) at position 39. The six other mutations at positions 226, 346,
436, 604, 682, and 925 being silent mutations.
The WOIRT gene from the P. mirabilis transconjugant differed from the WOTEM-2 gene
by one point mutation. This mutation consisted in the nucleotide change C-A at
position 929 which leads to the amino-acid substitution Arg-Ser at position 244
(Ambler et ah, 1991). This substitution has been previously described in IRT-2
(Belaaouaj et al., 1994) with pi 5.2 and derived from TEM-1 of pi 5.4. The same
decrease in pi value was observed in this IRT-2 with pi 5.4 derived from TEM-2 of
pi 5.6.
The specific activities of these IRT enzymes were proportionally lower than those
of their parent enzymes (Table II). The Km and K ^ values were similar whether
they originated from TEM-1 or TEM-2. The affinities (Km) of these IRT-enzymes
for benzylpenicillin, amoxycillin and ticarcillin were markedly lower than those of
TEM-1 and TEM-2 enzymes. After lOmin of incubation of the enzyme (0.5 U) in
presence of clavulanic acid at the concentration of 100 mg/L, a large excess of penicillin
G was added (1000 mg/L) and the enzymatic activity monitored. The initial activity
of IRT enzymes was 33% of that of a blank treated in the same conditions, but
without clavulanic acid. Enzymatic activity increased with time, following a
monomolecular kinetic with a time constant close to 0.01 sec"'. After 5 min the
enzymatic activity thus obtained was 90-95% of the initial value. These results are
consistent with the transient and fully reversible formation of an acyl-enzyme
with clavulanic acid. In the case of native TEM-1 and TEM-2 a similar acyl-enzyme
is also obtained and, if the concentration of clavulanic acid is low, reactivation
is also observed. However with the native enzymes, there is a progressive and irreversible
inactivation, whose mechanism is still unknown, and which does not occur with
IRTs.

256

1024
512
256
2

TEM-1
TEM-2
IRT2-2
IRT2-2
none

CF019

CF029
CF 1709
ATCC I0318IT
ATCC I03181T

Amoxycillin

Enzyme

Strain

32
256
128
2

+ clavulanate
(2 mg/L)

128

1024
32
4
1

Ticarcillin
8
4
2
0.5

+ clavulanate
(2 mg/L)
4
128
4
2
0.25

Piperacillin

Table I. MICs (mg/L) of /J-lactams for different P. mirabilis strains

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0.12
0.25
0.25
0.25
0.25

+ tazobactam
(4 mg/L)

8
8
2
4

Cephalothin
00

5
a
a.

330
400
300
300
30%
1700

100
140
2
1

375
360
460
312
33%
3120

100
145
2.3
2

IRT-2 derived
from TEM-2 (IRT : -2)

6100

27
35
11.5
600
9%

100
120
10.8
28

TEM-1

' im is expressed relative to benzylpenicillin as 100%.

Activity after lOmin of preincubation with clavulanic acid (CA) at concentration lOOmg/L.
'Specific activity is mU/mg.

/1 /

'Ka is fiM.

Benzylpcnicillin
Amoxycillin
Ticarcillin
Cephalothin
Residual activity of CA r
Specific activity*

IRT-2 derived
from TEM-l
*"
v

20
30
9
500
9%

10,000

100
120
10
25

TEM-2

Table II. Enzymatic properties of the /Mactamase IRT derived from TEM-2 compared with the /9-lactamase IRT-2 derived from
TEM-1 and with the parent enzymes TEM-1 and TEM-2

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188

L. Bret et al.

Acknowledgements

We thank Marlene Jan, Rolande Perroux and Dominique Rubio for their technical
assistance. This work was supported in part by a grant from the Direction de la Recherche
et des Etudes Doctorales, Ministere de TEducation Nationale, France.

References
Acar, J. C , Chardon H., Choutet, P., Courvalin, P., Dabernat, H., Drugeon, H. et al. (1995).
Antibiogram Committee of the French Society for Microbiology. 1995 Statement Pathologie
Biologie 43, I-VIII
Ambler, R. P., Coulson, A. F. N., Frere, J. M., Ghuysen, J. M., Joris, B., Forsman, M. eta!. (1991).
A standard numbering scheme for the class A /Mactamases. Biochemistry Journal 276,269-70.
Belaaouaj, A., Lapoumeroulie, C , Canica, M. M., Vedel, G., Nevot, P., Krishnamoorthy, R. et al.
(1994). Nucleotide sequences of the genes coding for the TEM-like /5-lactamases IRT-1 and
IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiology Letters 120, 75-80.
Bingen, E., Boissinot, C , Desjardins, P., Cave, H., Brahimi, N., Lambert-Zechovsky, N. et al.
(1993). Arbitrarily primed polymerase chain reaction provides rapid differentiation of Proteus
mirabilis isolates from a pediatric hospital. Journal of Clinical Microbiology 31, 1055-9.

Downloaded from http://jac.oxfordjournals.org/ by guest on February 26, 2012

The ten P. mirabilis strains isolated during the first six months of 1995 from different
patients in different wards were found to harbour the same IRT resistance phenotype.
Sequencing revealed the presence of the same IRT enzyme in all the strains. In addition
to the IRT enzyme, all strains except strain CHA produced a TEM enzyme of pi 5.4
as found in strain CF 1709.
RFLPs analysis (ribotyping) of the ten isolates showed that the same isolates
were grouped in six different ribotypes (A to F) (Table III) observed either with
EcoRI (Figure 1) or Hind III (Figure 2). Ribotype A included three strains
(DAU, PLA and SCI) isolated in three different units with the same resistance
to fluoroquinolones and aminoglycosides (Table III). Ribotype B included two
strains (BRO and LET) from two different units with the same resistance phenotype
(aminoglycoside resistance and fluoroquinolone susceptibility). Ribotype C included
two strains (PAH and SEU) isolated in the same unit with the same resistance
phenotype as in ribotype B. These last two strains probably came from the
same clone. Ribotype D differentiated one strain (CHA) only which was already
singular by its /?-lactamase content (IRT alone) and aminoglycoside resistance
phenotype (Table III). Ribotype E and F differentiated two strains (CF 1709 and
SER) from different units with the same resistance phenotype as in ribotypes B
and C.
Ribotyping results showed that these strains were genetically unrelated, which
rules out the epidemic dissemination of a clone. These findings suggest an
independent emergence of inhibitor resistance under antibiotic selective pressure in
P. mirabilis isolates as reported in Escherichia coli (Henquell et al., 1995).
TEM-2 is a frequently encountered penicillinase in P. mirabilis. Because the
prevalence of TEM-2 is higher in P. mirabilis (Philippon, Arlet & Lagrange, 1994)
than in K. pneumoniae and E. coli, it is not surprising that an IRT enzyme derived from
TEM-2 emerged in P. mirabilis. This is the first characterization of an IRT-2 enzyme
derived from TEM-2. We therefore suggest that this novel IRT enzyme be designated
IRT2-2.

neurology
neurology
Intensive Care Unit (ICU)
dermatology
pneumology
emergency
gastroenterology
dermatology
pediatric ICU
stomatology

DAU
BRO
LET
PAH
CHA
PLA
CF 1709
SEU
SCI
SER

urine
urine
wound
urine
pus
urine
pus
wound
urine
urine

Origin of
strain
A
B
B
C
D
A
E
C
A
F
A
F

E
C

A
B
B
C
D
A

Ribotype
EcoRI
Hind III

K, Kanamycin; G, gentamicin; T, tobramycin; NET, netilmicin; PEF, pefloxacin.

Unit of
isolation

Strain
(n = 10)

K-G-T-NET-PEF'
K-G-T-NET
K-G-T-NET
K-G-T-NET
K-G
K-G-T-NET-PEF
K-G-T-NET
K-G-T-NET
K-G-T-NET-PEF
K-G-T-NET

Associated resistance
markers

Table III. Origin, resistance phenotype and ribotype of the P. mirabilis strains studied

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190

L. Bret et al.

23.1 kb

9.4 kb

6.5 kb

4.3 kb

10

11

12

13

Figure 1. P. mirabilts rDNA RLFP nbotyping patterns obtained after EcoRI digestion. Lanes 1, 6, and
9: strains DAU, PLA, and SCI (Ribotype A); lanes 2 and 3: strains BRO and LET (Ribotype B); lanes 4
and 8: strains PAH and SEU f Ribotype C); lanes 5, 7 and 12- strains CHA (Ribotype D), CF 1709 (Ribotype
E) and SER (Ribotype F), lanes 10 and 11. strain CF 019, lane 13: strain ATCC 103181T.

4.3 kb

litliii

2.3 kb
2 kb

:S

7,

(i

,s

f)

10

11

12

13

Figure 2. P. nurahilis rDNA RLFP ribotyping patterns obtained after Hind III digestion. Lanes 1. 6. and
9: strains DAU. PLA, and SCI (Ribotype A), lanes 2 and 3 strains BRO and LET (Ribotype B); lanes 4
and 8 strains PAH and SEU (RibolypeC). lanes 5. 7 and 12. strains CHA (Ribotype D), CF 1709 (Ribotype
E) and SER (Ribotype F), lanes 10 and II. strain CF 019: lane 13: strain ATCC 103I81T.

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Inhibitor-resistant TEM in P. mirabitis

191

(Received 18 October 1995; returned 27 November 1995; revised 10 January 1996;

accepted 2 April 1996)

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Blazquez, J., Baquero, M. R., Canton, R., Alos, I. & Baquero, F. (1993). Characterization of a new
TEM-type /?-Iactamase resistant to clavulanate, sulbactam and tazobactam in a clinical isolate
of Escherichia coli. Antimicrobial Agents and Chemotherapy 37, 2059-63.
Bret, L., Romaszko, J. P., Henquell, C , Sirot, D., Chanal, C. & Sirot, J. (1994). Detection of
inhibitor-resistant TEM-/?-lactamases (IRT) by the automated ATB methods. \4th
Interdisciplinary Meeting on Anti-Infectious Chemotherapy, Paris, 1994. Abstract 323C16.
Brun, T., Pepduzzi, J., Canica, M. M., Paul, G., Nevot, P., Barthelemy, M. et al. (1994).
Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a
decreased susceptibility to /Mactamase inhibitors. FEMS Microbiology Letters 120, 111-117.
Chanal, C , Poupart, M. C , Sirot, D., Labia, R., Sirot, J. & Cluzel, R. (1992). Nucleotide sequences
of CAZ-2, CAZ-6 and CAZ-7 /J-lactamase genes. Antimicrobial Agents and Chemotherapy 36,
1817-20.
Chen, S. T. & Clowes, R. C. (1987). Variations between the nucleotide sequences of Tn\, Tnl and
Tn3 and expression of /?-lactamase in Pseudomonas aeruginosa and Escherichia coli. Journal of
Bacteriology 169, 913-6.
Goussard, S. & Courvalin, P. (1991). Sequence of the genes blaTAB and blaT-2. Gene 102,71-3.
Henquell, C , Chanal, C , Sirot, D., Labia, R. & Sirot, J. (1995). Molecular characterization of nine
different types of mutants among 107 inhibitor-resistant TEM /?-lactamases from clinical
isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 39, 427-30.
Labia, R., Andrillon, J. & Le Goffic, F. (1973). Computerized microacidimetric determination of
/Mactamase Michaelis-Menten constants. FEBS Letters 33, 42-4.
Lemozy, J., Sirot, D., Chanal, C , Hue, C , Labia, R., Dabernat, H. et al. (1995). First
characterization of inhibitor-resistant TEM (IRT) /Mactamases in Klebsiella pneumoniae
strains. Antimicrobial Agents and Chemotherapy 39, 2580-2.
Philippon, A., Arlet, G. & Lagrange, P. H. (1994). Origin and impact of plasmid-mediated
extended-spectrum beta-lactamases. European Journal of Clinical Microbiology and Infectious
Diseases 13, Suppl. I, 17-29.
Sirot, D., Chanal, C , Henquell, C , Labia, R., Sirot, J. & Cluzel, R. (1994). Clinical isolates of
Escherichia coli producing multiple TEM-mutants resistant to /Mactamase inhibitors. Journal
of Antimicrobial Chemotherapy 33, 111 7-26.
Zhou, X. Y., Bordon, F., Sirot, D., Kitzis, M. D. & Gutmann, L. (1994). Emergence of clinical
isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 /?-lactamase conferring
resistance to /Mactamase-inhibitors. Antimicrobial Agents and Chemotherapy 38, 1085-9.