Oxacillin-hydrolyzing β-lactamase involved in resistance to imipenem in Acinetobacter baumannii

FEMS Microbiology Letters 153 (1997) 333^339

Oxacillin-hydrolyzing L-lactamase involved in resistance to imipenem in Acinetobacter baumannii M. Hornstein a b , C. Sautjeau-Rostoker a c , J. Peèduzzi b , A. Vessieéres a , Le Thi Han Hong 1 a , M. Bartheèleèmy b , M. Scavizzi a *, R. Labia d ;

;

;

a

;

è riologie-Virologie, Centre Hospitalier Universitaire Avicenne, Universite è Paris Nord, 74 rue Marcel Cachin, Service de Bacte 93017 Bobigny Cedex, France

b

è um National d'Histoire Naturelle, Centre National de la Recherche Scienti¢que URA 401, 63 rue Bu¡on, 75231 Paris Cedex 05, France Muse

c

è Paris VI, 15 rue de l'Ecole de Me èdecine, Laboratoire de Recherche en Microbiologie sur les Antibiotiques, Universite 75270 Paris Cedex 06, France

d

è um National d'Histoire Naturelle, Centre National de la Recherche Scienti¢que, UMR 175, 6 rue de l'Universite è , 29000 Quimper, France Muse

Received 14 April 1997; accepted 2 June 1997

Abstract

31 ), synthesized two LAcinetobacter baumannii strain A148, a clinical isolate resistant to imipenem (MIC = 32 mg l lactamases with pIs 6.3 and 9.2. The pI 6.3 enzyme hydrolyzed the penicillins, including isoxazoylpenicillins, first-, secondand, to a lesser extent, third-generation cephalosporins. It was inhibited by chloride ions and by the penem L-lactamase inhibitor BRL 42715. Clavulanate was a weak inhibitor and EDTA did not affect the L-lactamase activity. This enzyme also hydrolyzed imipenem with a catalytic efficiency (kcat /Km ) of 1500 mM31 s31 . Moreover, this purified L-lactamase produced a positive microbiological clover-leaf test with imipenem. Therefore, the pI 6.3 L-lactamase was considered to be involved in the imipenem resistance of A. baumannii strain A148.

s

Keywords :

Imipenem resistance;

Acinetobacter

; Oxacillin-hydrolyzing L-lactamase

1. Introduction

Acinetobacter baumannii is an important pathogen in nosocomial infections. There has been a noticeable increase in the frequency of this species in recent years with high mortality or, at the very least, pro-

* Corresponding author. Service de Bacteèriologie-Virologie, Hoêpital Avicenne, 125 rue de Stalingrad, 93009 Bobigny Cedex, France. Tel.: (33) (1) 48 95 56 11. Fax: (33) (1) 48 95 59 11. 1 Present address: Institut National d'Hygieéne and d'Epide¨miologie, 1 rue Yersin, Hanoi, Vietnam.

longed stays in intensive care units. This is partly due to its resistance to L-lactam antibiotics. A. baumannii often synthesizes a chromosomally mediated cephalosporinase [1,2] belonging to group 1 according to the classi¢cation of Bush et al. [3]. Some strains also produce TEM-type penicillinases [1,4,5]. A carbenicillin-hydrolyzing L-lactamase CARB 5 [1,6] and an extended-spectrum L-lactamase (ESBL) [7] were also described in this species. In addition, A. baumannii has developed an intensive pattern of resistance to other antibiotic families and when this was the case, imipenem was the only available molecule for the

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 7 0 - X

FEMSLE 7698 20-10-97

M. Hornstein et al. / FEMS Microbiology Letters 153 (1997) 333^339

334

treatment

of

A. baumannii

such

infections.

strains

have

Imipenem-resistant

recently

been

detected

nem disk was placed in the center of the plate. The di¡erent

strains

to be tested were

smeared out in

[8,9] and outbreaks of such resistance have been re-

streaks radially from the disk. The plates were incu-

ported [1,10].

bated overnight at 37³C. With the

The

A. baumannii

strain A148, isolated in our hos-

ducing

strains,

hydrolyzing

L-lactam anti-

streaks,

M. luteus

biotics including carbapenems. The aim of this paper

thereby

inducing

pital, was highly resistant to all tested

was to test the possible relation between the

L-lac-

zone

was

able

by

indentations

M. luteus

of

L-lactamase-pro-

imipenem

inhibition.

around

itself

to

inside

the

Strains

the

develop, circular

A148

and

tamase content of this strain and its resistance phe-

A145 were comparatively tested as well as positive

notype.

and negative controls. A variation of this test was to

2. Materials and methods

I 6.3 L-lactamase puri¢ed from an extract of A. baumannii A148, and the pI 9.2 L-lactamase from A. baumannii A145. Both were poured into use the p

s

holes at a distance of 2 cm from a central imipenem

2.1. Bacterial strains

disk. Conjugation

A. baumannii

A148,

resistant

to

imipenem,

was

experiments

between A148 and

E. coli A. junii

were

attempted

¢rstly

strains at 37³C and then

isolated in December 1989 from the urine of a 91

between A148 and

year old man in the Gastroenterology Department

according to the method described in [13]. No anti-

ê pital Avicenne, Bobigny, France. The identi¢of Ho

biotic was available to select recombinants from do-

cation of the strain was con¢rmed by API strip NE

nor cells because

è rieux SA, Marcy l'Etoile, France) and by cul(Biome

ant

ture on trypticase soy (TS) agar plates (Sano¢ Diag-

azide). Therefore (i) a metabolic character (lactose)

(including

strains at 25³C and 30³C

A. baumannii

rifampicin,

E. coli

A148 was multiresist-

minocyclin

and

sodium

nostics Pasteur SA, Marne-la-Coquette, France) in-

was used to detect

cubated at 44³C.

ski agar plates and (ii) the morphology of the colo-

A. baumannii hospital,

comparison. (kindly

strain

susceptible

A145,

to

isolated

imipenem,

Acinetobacter junii

provided

by

P.

in

the

was

same

used

for

strain ATCC 17908

Bouvet,

Institut

transconjugants on Drigal-

nies made it possible to detect the jugants

on

trypticase

soy

A. junii

agar.

transcon-

Imipenem

was

included in agar for both experiments.

Pasteur,

Assays to cure the imipenem resistance character

Paris) was used as negative control for the micro-

were carried out with 5 mM ethidium bromide [14].

A. baumannii

biological clover-leaf test. An imipenem-hydrolyzing

L-lactamase-producing maltophilia,

strain

of

Stenotrophomonas

taken from our collection, was chosen

as positive control. An imipenem-sensitive

cus luteus were

Micrococ-

strain was used in the test. Two strains

assayed

as

recipients

Escherichia coli A. junii. ments :

in

conjugation

experi-

DNA

A148 was examined for its plasmid

content by the Birnboim and Doly method

[15].

2.3. Analytical isoelectric focusing (IEF) and L-lactamase puri¢cation

IP J 53-2 and the previous Bacterial cultures were developed in trypticase soy broth. Analytical isoelectric focusing was performed

2.2. Antibiotic susceptibility assay. Microbiological clover-leaf test and genetic studies

with crude enzyme extracts as previously described [16] on polyacrylamide gels containing ampholytes

I

with a pH range of 3.5^9.5. Their p s were measured The were

minimum

determined

inhibitory by

the

concentrations

macrodilution

(MICs)

method

in

Mu ë ller-Hinton broth [11]. Microbiological

clover-leaf

I

tests

were

performed

M. luteus.

One imipe-

L-lactamase extracts of known

p : TEM-1 (5.4), TEM-2 (5.6), TEM-3 (6.3), SHV-1 (7.7),

according to [12]. A Mu ë ller-Hinton agar plate was £ooded with a suspension of

by a comparison with

CAZ-4/SHV-5

AmpC

(9.2).

(8.2),

Puri¢cation

and of

E. coli K12 RL90 I 6.3 L-lactamase

p

from crude extract of strain A148 by size exclusion chromatography on Sephadex G-150 and by prepar-

FEMSLE 7698 20-10-97

335

M. Hornstein et al. / FEMS Microbiology Letters 153 (1997) 333^339

Table 1 L-Lactam antibiotic MICs for A. baumannii strains A145 and A148 Antibiotic MIC (mg l31 ) A145 A148 Amoxycillin 32 s 256 16 s 256 Amoxycillin+clavua Ticarcillin 256 s 512 Ticarcillin+clavua 128 512 Piperacillin 8 128 Cephalothin 256 s 256 Cefoxitin 128 s 256 Cefotaxime 64 128 Ceftazidime 32 s 256 Cefpirome 64 128 Meropenem 0.125 32 Imipenem 0.25 32 Imipenem+BRL 42715 b ^ 0.5 a Clavulanate 2 mg l31 . b BRL 42715 1 mg l31 .

ative isoelectric focusing was performed as previously described [17]. The homogeneity and the relative molecular mass of the puri¢ed enzyme were determined by analytical SDS-PAGE as described by Laemmli [18]. The molecular mass calibration kit Dalton Mark VII-L (Sigma, Saint Quentin Fallavier, France) was used. 2.4. Substrate and inhibitor pro¢les

One unit of L-lactamase activity was de¢ned as the amount of enzyme hydrolyzing 1 Wmol of benzylpenicillin per minute at 37³C and pH 7. During the puri¢cation procedure of the L-lactamase and the inhibition study with p-hydroxymercuribenzoate (PHMB) and EDTA, the enzymatic activity was checked spectrophotometrically at 235 nm in 50 mM sodium phosphate bu¡er, at pH 7, with 800 WM benzylpenicillin as substrate. Kinetic parameters (kcat and Km ) of the puri¢ed pI

6.3 L-lactamase from strain A148 were determined for the substrates listed in Table 1, at 37³C by computerized microacidimetry [19] in 0.8 WM bovine serum albumin. Hydrolysis of 60 WM imipenem was followed spectrophotometrically at 300 nm because the microacidimetric method was at the limit of sensitivity. For substrate hydrolysis, catalytic e¤ciency was de¢ned as the ratio kcat /Km . The Ki value of the inhibitors or poor substrates was measured using a competition procedure with 600 WM benzylpenicillin as substrate. For the inhibition by PHMB, EDTA, NaCl, clavulanate, tazobactam and sulbactam, several concentrations of each inhibitor were preincubated with the enzyme for 5 min before the remaining activity was measured. Thus the inhibitor concentration required to inhibit 50% of the enzyme activity (IC50) was determined. Vmax and Km of the pI s 9.2 L-lactamase were determined by the microacidimetric method with crude extracts of strains A145 and A148, the latter being previously brought to 140 mM NaCl to inhibit the activity of the pI 6.3 oxacillin-hydrolyzing L-lactamase. 3. Results

3.1. MICs

L-Lactam antibiotic MICs for strains A148 and A145 are shown in Table 1. Unlike A145, an unusually high imipenem MIC was observed for A148. The penem L-lactamase inhibitor BRL 42715 [23] restored the susceptibility of strain A148 to imipenem. Moreover, the MICs of amoxycillin and piperacillin were clearly higher for A148. 3.2. Analytical isoelectric focusing

Analytical IEF of crude extract from strain A148

Table 2 Puri¢cation of pI 6.3 L-lactamase from A. baumannii A148 Procedure Total activity (U) a Crude extract 674 Gel ¢ltration on Sephadex G 150 632 Preparative isoelectric focusing 110 a Obtained from 30 g wet bacteria.

FEMSLE 7698 20-10-97

Speci¢c activity (U mg31 ) 0.35 3.4 275

Yield (%) ^ 94 16

336

M. Hornstein et al. / FEMS Microbiology Letters 153 (1997) 333^339

latter was responsible for the imipenem inactivation (Fig. 2). 3.5. Imipenem resistance transfer. Plasmid DNA content

Attempts to transfer the imipenem resistance character by conjugation from strain A148 to recipient strains were unsuccessful. Ethidium bromide treatment did not cure the imipenem resistance character in strain A148. Plasmid DNA could not be detected with the method of Birnboim and Doly [15]. 3.6. Substrate and inhibitor pro¢les

Fig. 1. Clover-leaf test results for A. baumannii A148 and A145 and controls. A: A. baumannii A148; B: A. baumannii A145; C: A. junii (negative control); D: S. maltophilia (positive control); E: imipenem disk; F: M. luteus culture.

revealed two main spots with L-lactamase activity at pIs 6.3 and s 9.2, besides two minor spots at pIs 6.7 and 7.1. Extract of strain A145 exhibited a single band at pI s 9.2 which co-migrated with that of strain A148 (results not shown).

The pI 6.3 L-lactamase from A. baumannii A148 showed a high hydrolytic activity against penicillins and ¢rst-generation cephalosporins, whereas values of kcat for third-generation cephalosporins, monobactam and carbapenems were low (Table 3). Isoxazoylpenicillins, especially oxacillin, were the best substrates. The kcat value for imipenem was low. However, as this carbapenem had a high a¤nity (Ki : 0.4 WM), it was as good a substrate as isoxazoylpenicillins in terms of catalytic e¤ciency.

3.3. L-Lactamase puri¢cation and relative molecular mass determination

The two-step procedure for the puri¢cation of the pI 6.3 L-lactamase from strain A148 crude extract is shown in Table 2. The yield of preparative IEF was rather low (17.5%) whereas the L-lactamase was puri¢ed 80-fold in this single step. SDS-PAGE analysis indicated a purity of 95% and an apparent molecular mass of 28 000. Bands with minor L-lactamase activity that focused at pIs 6.7 and 7.1 were also collected in order to determine their substrate pro¢les. 3.4. Agar plate microbiological clover-leaf test

Fig. 1 shows a comparative clover-leaf test with imipenem for A. baumannii strains A148 and A145. The test was positive with strain A148. Moreover, the test using crude extract of strain A145 and the puri¢ed A148 pI 6.3 L-lactamase indicated that the

Fig. 2. Microbiological clover-leaf test results for L-lactamase extracts from A. baumannii A148 and A145 and controls. A: pI 6.3 L-lactamase of A. baumannii A148; B: pI s 9.2 L-lactamase of A. baumannii A145; C: distilled water (negative control); D: crude extract from S. maltophilia (positive control); E: imipenem disk; F: M. luteus culture.

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M. Hornstein et al. / FEMS Microbiology Letters 153 (1997) 333^339

Table 3 Kinetic properties of pI 6.3 L-lactamase from A. baumannii A148 Km kcat /Km Antibiotic kcat (s31 ) (WM) (mM31 s31 ) Benzylpenicillin 120 5 24 000 Amoxycillin 90 14 6 400 Ticarcillin 16 8 1 950 Oxacillin 192 76 2 500 Cloxacillin 115 143 800 Flucloxacillin 162 124 1 300 Cephalothin 99 112 880 Cephaloridine 114 57 2 000 Cefoxitina 6 0.4 49 6 10 Cefotaximea 3.6 210 20 Ceftazidimea 6 0.4 s 500 6 10 Aztreonama 2.4 s 500 6 10 Imipenema 0.6 0.4 1 500 Meropenema 1.2 1.25 960 BRL 42715a NDb 0.08 ^ Clavulanatea ND 2 ^ Tazobactama ND 39 ^ ND 453 ^ Sulbactama a Km values were determined as Ki by substrate (benzylpenicillin) competition. b ND: not determined.

PHMB (100 WM) and EDTA (1 mM) did not alter the pI 6.3 L-lactamase activity which was weakly inhibited by clavulanate whereas tazobactam and sulbactam were poor inhibitors. Conversely, the enzyme was susceptible to inhibition by chloride ions: IC50 of NaCl was 6 mM. In the presence of 140 mM NaCl, less than 1% of the L-lactamase activity remained. On the basis of substrate and inhibitor pro¢les, the pI 6.3 oxacillin-hydrolyzing L-lactamase from strain A148 belongs to group 2d of the Bush et al. classi¢cation. [3]. Amongst those L-lactamases focusing at pIs 6.7 and 7.1, the relative hydrolysis values obtained with benzylpenicillin, oxacillin, cefotaxime and imipenem were not signi¢cantly di¡erent from those of the major L-lactamase (pI 6.3) (data not shown). This suggests that they are isoenzymes. Kinetic constants of the pI s 9.2 L-lactamase from strain A148 were determined using crude extracts after inhibition of the oxacillin-hydrolyzing enzyme with 140 mM NaCl. Data for Km and relative Vmax are shown in Table 4. Strain A145 also produced a L-lactamase of pI s 9.2. Substrate pro¢les for both enzymes were similar (data not shown). The L-lactamases had a high a¤nity for all L-lactams

337

tested except for ¢rst-generation cephalosporins. Conversely, the latter were the only antibiotics which were well hydrolyzed. Moreover, the enzymes did not interact with cefpirome and clavulanate. The speci¢c activities (using cephaloridine as substrate) of L-lactamases of pI s 9.2 from strains A145 and A148 were 0.9 and 0.05 U mg31 respectively. Therefore, the production of these enzymes can be considered to have no impact on the susceptibility of A. baumannii strains to third-generation cephalosporins, monobactam and carbapenem. 4. Discussion

Generally, the resistance of A. baumannii to imipenem is the result of alteration in the expression of penicillin binding [20], or outer membrane proteins [21]. Two cases of imipenem resistance, however, have been found to be associated with the presence of L-lactamase: AbRC-1 [8] and ARI-1 [9]. In the latter, imipenem resistance could be transferred to A. junii by conjugation and a plasmid of 45 kb named pUK1356 could be identi¢ed in the transconjugant [22]. In our study, A. baumannii strain A148 exhibited imipenem resistance (MIC=32 mg l31 ). A positive clover-leaf test was achieved with imipenem and Table 4 Substrate pro¢le of pI s 9.2 L-lactamase from A. baumannii A148 Vmax a Antibiotic Km (WM) b Benzylpenicillin 0.15 25 Ticarcillin 0.67b 5 3.5 Piperacillin 0.57b Cephaloridine 500 100 Cephalothin 375 200 Cefotaxime 0.25b NHc Cefpirome NId NI Ceftazidime 3.3b NH Aztreonam 0.7b NH NH Imipenem 3b Clavulanate NI NI a Data are given as percentage of the cephaloridine value. b Km values were determined as Ki by substrate (cephaloridine) competition. c NH: no hydrolysis was detected. d NI: no interaction was found.

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M. Hornstein et al. / FEMS Microbiology Letters 153 (1997) 333^339

338

the susceptibility to this antibiotic was restored by

L-lactamase

the penem

results are in agreement with an enzymatic imipenem resistance

mechanism

in

this

strain.

s

I

L-lactamase which,

9.2

A. baumannii

L-lactamases :

A148 was shown to produce two p

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I

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6.3. The

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