Ceftibuten stability to active-site serine and metallo-ß-lactamases

International Journal of Antimicrobial Agents 17 (2001) 45 – 50 www.ischemo.org

Original article

Ceftibuten stability to active-site serine and metallo-ß-lactamases Mariagrazia Perilli a, Bernardetta Segatore a, Nicola Franceschini a, Giovanni Gizzi a, Andrea Mancinelli a, Berardo Caravelli a, Domenico Setacci a, Maria del Mar Tavio-Perez a,b, Bruno Bianchi c, Gianfranco Amicosante a,* a

Department of Sciences and Biomedical Technologies, School of Medicine, Uni6ersity of L’Aquila, Via Vetoio, 67010 Coppito L’ Aquila, Italy b Department of Clinical Sciences, Uni6ersity of Gran Canaria, Las Palmas, Gran Canaria, Spain c Medical Department, Schering Plough, Milan, Italy Received 25 May 2000; accepted 19 July 2000

Abstract Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several ß-lactamases because of the carboxyethilidine moiety at position 7 of the ß-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ß-lactamases were investigated. The activity of several TEM-derived extended spectrum ß-lactamases (ESßLs) against ceftibuten, cefotaxime and ceftazidime was compared using Km, Kcat and Kcat/Km. Ceftibuten behaved as a poor substrate for class A and B ß-lactamases compared with cefotaxime. The chromosomal class C ß-lactamase from Enterobacter cloacae 908R gave a high Kcat value (21 s − 1), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing ß-lactamases. © 2001 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Ceftibuten; ß-Lactamases; Affinity; Catalytic efficiency

1. Introduction Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria, including Enterobacteriaceae, Neisseria spp., Moraxella catarrhalis, Haemophilus influenzae, ß-haemolytic streptococci and penicillin-susceptible Streptococcus pneumoniae [1]. Ceftibuten shows an improved stability to hydrolysis by several ß-lactamases because of the carboxyethilidine moiety at position 7 of the ß-acyl side chain. As reported by some authors, strains of Escherichia coli producing TEM-1, TEM-2 and the extended spectrum derivatives TEM-3, TEM-4, TEM-5, TEM-6, TEM-7 and TEM-9 were sensitive and showed MIC values 5 8 mg/l. The same behaviour was reported with strains producing SHV-1, SHV-2 and OXA-1, OXA-2 and OXA-3 enzymes (MIC values 5 4 mg/l) * Corresponding author. Tel.: +39-862-433455; fax: + 39-862433433. E-mail address: [email protected] (G. Amicosante).

[2–5]. Moreover, strains of Klebsiella pneumoniae producing SHV-4 or SHV-5 were less susceptible to ceftibuten if compared with K. pneumoniae strains producing SHV-1, SHV-2, SHV-3, TEM-1 and K1 ß-lactamases [2,6,7]. The stability of ceftibuten reported against ESBLs makes this compound more active than or similar to the in vitro activity of some injectable third-generation cephalosporins against Enterobacteriaceae producing the plasmid-encoded extended spectrum enzymes listed above [1]. Ceftibuten has been reported to be poorly hydrolysed by chromosomal cephalosporinase from Enterobacter cloacae (P99 enzyme) [8] and inactivated by CARB enzymes produced by Pseudomonas aeruginosa [4]. The compound is also hydrolysed by the plasmid-encoded ß-lactamases, CMY-1 and MIR-1, produced by clinical isolates of K. pneumoniae [2]. Ceftibuten is used in therapy for the treatment of urogenital and respiratorytract infections in both adults and children. Such infections could be caused by organisms producing either active-site serine or metallo ß-lactamases.

0924-8579/01/$ - $20 © 2001 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 0 ) 0 0 3 1 9 - 8

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The present study was focussed on the interaction between ceftibuten with a wide range of ß-lactamases in order to determine either the stability to hydrolysis (kcat or Vmax) or the affinity (Km or Ki) for this compound as major determinant of resistance. The measurement of those parameters could help rationalise the in vitro-microbiological activity of ceftibuten against strains producing these enzymes.

2. Materials and methods Chemicals used were the purest analytical grade obtained from commercial sources. Culture media and nitrocefin were from Unipath, Milan, Italy. Ceftibuten was supplied by Schering-Plough (Milan, Italy). The strains used in this study belonged to the collection of the University of L’Aquila. Nineteen enzymes were studied. In Table 1, the enzymes are assigned to molecular class A, B and C using Ambler’s classification [9]. All the b-lactamases (except the enzyme produced by Acinetobacter baumanni TR187) have been sequenced. The BlaIMP1 enzyme from E. coli BL21 was kindly provided by Dr M. Galleni (CIP, Lie`ge, Belgium). Table 1 ß-Lactamases used in the present study Enzyme source

Molecular class and locationa

pI

TEM-1, E. coli

Class A-ESbL plasmid mediated Class A-EbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-ESbL plasmid mediated Class A-chromosomal Class B-chromosomal Class B-chromosomal

5.4

TEM43, M. morganii TEM-52, P. mirabilis TEM-60, E. coli TEM-AQ, E. coli TEM-72, P. mirabilis SHV-1, E. coli SHV-2a, E. coli SHV-5, E. coli SHV-12, E. coli ULA-27, C. di6ersus VIM-1, P. aeruginosa BlA-B, C. meningosepticum BlAimpl, P. aeruginosa BC II, Bacillus cereus AE036, A. hydrophila TR187, A. baumannii 908 R, E. cloacae MM-89, M. morganii a

Class Class Class Class Class Class

B-plasmid mediated B-chromosomal B-chromosomal C-chromosomal C-chromosomal C-chromosomal

According to the Ambler’s scheme [9].

6.1 6.0 6.4 5.5 5.9 7.6 7.6 8.2 8.2 6.8 5.1 8.5 8.0 8.3 8.0 \9 \8 7.7

Seventeen b-lactamases were highly purified (]95%); as SHV-2a and SHV-12 were less pure preparations (around 80%), only Vmax values were calculated. The enzymes from E. coli (TEM-AQ) [10], Pro6idencia stuartii (TEM-60) [11], Citrobacter di6ersus-ULA27 [12,13], Chryseobacterium meningosepticum BLA-B [14], Bacillus cereus BC-II [15], Aeromonas hydrophyla AE036 [15], A. baumanii TR-187 [16] and E. cloacae 908-R [17] were purified, as reported previously. The enzymes from Proteus mirabilis (TEM-52 and TEM-72) and P. aeruginosa VIM-1 were purified, as reported elsewhere (manuscript submitted). The ß-lactamases produced by E. coli (TEM-1, SHV2a and SHV-12) and Morganella morganii TEM-43 were purified as follows. Bacteria were grown in 4 l of brain-heart-infusion (BHI) medium for 18 h at 37°C under orbital shaking (180 rpm) and harvested by centrifugation at 8000× g for 20 min at 4°C. The pellet was washed twice with 30 mM Tris HCl buffer pH 8.0 or with 50 mM sodium phosphate buffer pH 6.7 for TEM and SHV enzymes, respectively. The cells were disrupted by cycles of ultrasonic treatment (five times for 30 s at 60 W each time). Crude extracts were centrifuged at 105 000× g for 30 min and the cleared supernatants were recovered and loaded onto a Sepharose-Q fast-flow column (XK 26/20, AmershamPharmacia Biotech, Milan, Italy) equilibrated with 50 mM Tris–HCl buffer pH 8.0 or onto a Sepharose-S fast flow (XK 26/20, Amersham-Pharmacia Biotech, Milan, Italy) equilibrated with 50 mM sodium phosphate buffer pH 6.7 for TEM and SHV enzymes, respectively. The b-lactamases were eluted over a linear gradient of NaCl (0.2–0.8 M) for 80 min at a flow rate of 3 ml/min. The active fractions were pooled and loaded onto a Superdex 75 (XK 26/70, AmershamPharmacia Biotech, Milan, Italy) equilibrated with 50 mM sodium phosphate buffer pH 7.0. Each TEM-type b-lactamase was purified as follows. The fractions containing the enzyme activity were pooled, dialysed overnight at 4°C against 25 mM BisTris buffer pH 7 and loaded onto a Mono P HR 5/20 column (Amersham-Pharmacia Biotech, Milan, Italy) equilibrated with the same buffer. The proteins were eluted with 25 ml of 10-fold diluted polybuffer 74 (range 7–4). The enzyme purity was determined by sodium dodecilsulphate polyacrylamide gel electrophoresis (SDSPAGE) analysis performed by the method of Laemmli [18] and protein content was determined by the method of Bradford [19] with bovine-serum albumin as the standard. The hydrolysis of ceftibuten was followed by a spectrophotometric assay. An absorbance spectrum of 100 mM ceftibuten before and after complete hydrolysis was recorded in order to determine the wavelength and the molar extinction coefficient value at 30°C (l, 260 nm;

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Table 2 Interaction of ceftibuten with class A ß-lactamasesc Enzyme

TEM-1 TEM-AQ TEM-43 TEM-52 TEM-60 TEM-72

Cefotaxime†a

Ceftibuten

Ceftazidime†a

Km (mM)

kcat (s−1)

kcat/Km (mM/s)

Km (mM)

kcat (s−1)

kcat/Km (mM/s)

Km (mM)

kcat (s−1)

kcat/Km (mM/s)

800*b 340 963 313 909 435

N.H.**b 11 10 20 8 4

N.D.***b 32×10−3 10×10−3 64×10−3 9×10−3 9×10−3

6000 190 33 15 31 22

9 0.3 6.9 170 1.5 3.7

1.5×10−3 1.6×10−3 210×10−3 11 50×10−3 168×10−3

4280 115 160 166 59 75

0.3 0.4 43 10 12 0.6

7×10−5 3×10−3 270×10−3 60×10−3 200×10−3 8×10−3

a

†, The kinetics data from TEM-1, TEM-AQ, TEM-43, TEM-60 relative to cefotaxime and ceftazidime were taken from the references [10,11,22,23], respectively. For TEM-52 and TEM-72, source is on manuscripts submitted for pubblication). b *, Km was determined as Ki using 130 mM nitrocefin as substrate reporter; **, N.H., not significant hydrolysis; ***, N.D., not determined. c TEM-derived. Table 3 Interaction of ceftibuten with class A ß-lactamasesa Enzyme

Km (mM)

kcat (s−1)

kcat/Km (mM/s)

Vmax (moles min/mg)

SHV-1 (E. coli ) SHV-5 (K. pneumoniae) SHV-2a§b(E. coli ) SHV-12§b(E. cloacae)

400*b 1000 277 100

N.H.**b 10

N.D.***b 0.01 N.D. N.D.

1.8×10−6 7.2×10−6

a

SHV-derived. *Km was determined as Ki using 130 mM nitrocefin as substrate reporter; **, N.H., not significant hydrolysis; ***, N.D., not determined; §, partially purified. b

recorded in order to determine the wavelength and the molar extinction coefficient value at 30°C (l, 260 nm; −1 Do 260 cm − 1). The complete ceftibuten M , − 3200 M hydrolysis was performed by using 5 mg of pure BlaIMP1 ß-lactamase. Ceftibuten hydrolysis in the presence of each ß-lactamase was followed at 260 nm at 30°C in 50 mM phosphate buffer pH 7.0 for classes A and C. The metallo-ß-lactamases used in this study were assayed in 30 mM Hepes buffer pH 7.2 added with 50 mM ZnCl2. Measurements were performed at 30°C. Enzyme preparations below 0.1 mg/ml were added with 50 mg/ml of BSA. The metallo-ß-lactamase activity from A. hydrophila AE036 was assayed in 30 mM sodium cacodylate buffer without zinc addition. The kinetic parameters at the steady-state (Km and kcat) were determined by measuring the initial rate of hydrolysis of the ceftibuten, as previously reported. Calculations were made by the Hanes-Woolf plot [20]. When the Km for ceftibuten was under 60 – 70 mM, a Ki value was calculated using the substrate reporter method with 130 mM nitrocefin as substrate.

3. Results Ceftibuten hydrolysis was monitored in the presence

of five TEM-derived ESBLs. The kinetic parameters were compared with those obtained for two broad spectrum injectable cephalosporines, cefotaxime and ceftazidime (Table 2). Ceftibuten had less affinity than cefotaxime and ceftazidime and had Km values several times higher. TEM1 enzyme hydrolysed cefotaxime and, to a lesser extent, ceftazidime, but did not inactivate ceftibuten under our experimental conditions. Ceftibuten catalytic efficiencies (kcat/Km) were always less than those obtained for cefotaxime and ceftazidime with TEM-43, TEM-52, TEM-60 and TEM-72 but TEM-AQ inactivated ceftibuten 10–20 times more than either of the two cephalosporines. SHV-5, SHV-2a and SHV-12 enzymes were able to hydrolyse ceftibuten with the exception of SHV-1. However, as reported in Table 3, SHV-5 showed Km values of about 1 mM, with a catalytic efficiency kcat/ Km of 10 − 2 mM/s; these were very similar to those calculated for TEM-derived enzymes (Table 2). SHV-2a and SHV-12 ß-lactamases showed greater affinity compared with SHV-1 and SHV-5 enzymes. SHV-12 enzyme gave a Vmax value (expressed as moles min/mg) four times higher than SHV-2a. Table 4 lists the kinetic parameters for the interaction of ceftibuten with some metallo-ß-lactamases. Ceftibuten was a good substrate for all the ß-lactamases

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tested. The only exception was the enzyme produced from A. hydrophila AE036 that was not able to hydrolyse the antibiotic; no inhibition of enzyme activity was seen during incubation in the presence of 1 mM ceftibuten and a reporter substrate. B. cereus and IMP1 enzymes showed a catalytic efficiency (kcat/Km) 3–4fold lower than those reported for cefotaxime. Surprisingly, with the metallo-ß-lactamase from C. meningosepticum, the value of catalytic efficiency fell to 70-fold lower. The activity of VIM-1 and IMP-1 enzymes with ceftazidime (Franceschini et al., manuscript submitted for publication; [11]) was different because the catalytic efficiency calculated for ceftibuten was 2 – 8-fold higher than ceftazidime. In the present study, we compared three class C ß-lactamases with a chromosomal class A enzyme from C. di6ersus ULA-27 [12,13] (Table 5). C. di6ersus ULA27 enzyme effectively inactivated ceftibuten with a catalytic efficiency similar to TEM- and SHV-derived ß-lactamases. Surprisingly, E. cloacae 908-R cephalosporinase (similar to P99 enzyme) hydrolysed ceftibuten with a kcat value similar to that of TEM-52 and TEM-AQ ESBLs. Usually, class C ß-lactamases show a high affinity (low Km) for third-generation cephalosporins [21], but the Km value for ceftibuten of E. cloacae 908-R enzyme was exceptionally high (Km = 654 mM). A. baumannii TR187 and M. morganii MM89 enzymes showed greater affinity. However, in our experimental conditions, both cephalosporinases did not hydrolyse ceftibuten which behaved as very poor substrate with very low kcat values.

4. Discussion Ceftibuten is an oral, third-generation cephalosporin active in vitro against most of Enterobacteriaceae and some bacterial species involved in respiratory tract infections, such as M. catarrhalis and H. influenzae, all of which are able to produce different types of ß-lactamases. Ceftibuten is used for the treatment of urinary, respiratory and genital tract infections, some of which could be caused by bacteria producing either active-site serine or rarely metallo-ß-lactamases. Several reports have been published on ceftibuten stability to ß-lactamases, such as TEM-1, OXA-1, SHV-1, CARB-2, P99 or to broad-spectrum enzymes, such as TEM-3, SHV-4, SHV-5 [24]. Moreover, Medeiros and Crellin [25], evaluated ceftibuten activity against bacteria producing ESBLs in the presence of large inocula. Strains producing SHV-4 and SHV-5 ß-lactamases had increased MIC values. Starting from the published data, because of this high stability to hydrolysis, ceftibuten is useful against a wide range of ß-lactamase-producing bacteria. In this study, we have evaluated ceftibuten stability, investigating the kinetic parameters in detail against a wide array of active-site serine and metallo-ß-lactamases. We confirmed that TEM-1 and SHV-1 ß-lactamases were unable to hydrolyse the compound. One of the major differences in hydrolytic stability was seen with a cephalosporinase from E. cloacae (908R) whose nucleotide sequence is quite similar to the enzyme named, P99. We found that 908R enzyme was able to inactivate ceftibuten at a significant rate. Moreover, the catalytic

Table 4 Interaction of ceftibuten with class B ß-lactamases Enzyme

Bla-B VIM-1 B. cereus IMP-1 a

Cefotaximea

Ceftibuten Km (mM)

kcat (s−1)

kcat/Km (mM/s)

Km (mM)

kcat (s−1)

kcat/Km (mM/s)

260 422 423 135

0.8 80 57 189

3.1×10−3 190×10−3 140×10−3 1.4

180 247 90 4

39 169 60 1.3

220×10−3 680×10−3 670×10−3 350×10−3

The data were taken from the references [14,15]. For VIM-1 enzyme, data are on the manuscript submitted for publication.

Table 5 Interaction of ceftibuten with chromosomal ß-lactamasesa Enzyme

Molecular class

Km (mM)

Kcat (s−1)

kcat/Km (mM/s)

908R (E. cloacae) TR187 (A. baumannii ) MM-89 (M. morganii ) ULA-27 (C. di6ersus)

C C C A

654 1.4* 0.38* 200

21 4×10−3 1.6×10−3 23

32×10−3 2.8×10−3 4.2×10−3 115×10−3

a

*, Km was determined as Ki, using 130 mM nitrocefin as substrate reporter.

M. Perilli et al. / International Journal of Antimicrobial Agents 17 (2001) 45–50

efficiencies obtained with the cephalosporinases from A. baumannii TR187 and M. morganii MM89 against ceftibuten were significant. Despite the promising kinetics, the high ceftibuten MIC values found in strains overproducing AmpC is due to the high concentration reached by these enzymes in the periplasmic space. Concerning the SHV-type ESBL, we confirmed that SHV-5 hydrolysed the compound efficiently, resulting in resistance; in addition, SHV-2a and SHV-12 inactivated ceftibuten, with SHV-12 being more efficient than SHV-2a. The interaction of ceftibuten with TEM-type ESBL was quite interesting. If we compared the catalytic efficiency of ceftibuten, cefotaxime and ceftazidime with TEM-AQ, TEM-43, TEM-52, TEM-60 and TEM-72, we found ceftibuten was generally more stable than both cefotaxime and ceftazidime with the exception of TEMAQ, which is a complex TEM mutant [10]. Ceftibuten had the higher Km values, indicating a diminished affinity for the TEM-ESBL derivatives, most probably due to the different side chain of ceftibuten. We also looked at the behaviour of ceftibuten against five metallo ß-lactamases. We confirmed that A. hydrophila AE036 is unable to hydrolyse ceftibuten because of the enzyme’s very narrow spectrum of activity. The more active metallo-enzyme against the compound was the Bla-Imp-1 ß-lactamase produced by P. aeruginosa. The enzyme is also produced by some Enterobacteriaceae; spread is via a plasmid [26]. Ceftibuten was, however, hydrolysed at a lesser extent compared with cefotaxime, confirming its good stability even in the presence of such extended-spectrum metallo ß-lactamases. Moreover, with these enzymes, the Km values observed for ceftibuten were also higher than those reported for cefotaxime. Due to the heterogeneity of active-site serine and metallo-ß-lactamases, all ß-lactamases are hydrolysed by these enzymes, inhibitors included. Moreover, our data indicated ceftibuten is similarly or more resistant to hydrolysis when compared with some injectable thirdgeneration cephalosporins. It is reasonable to think that in the presence of respiratory or urogenital tract-bacterial flora producing typical ß-lactamases at low levels of production, ceftibuten will maintain stability to a great extent and resist hydrolysis by these enzymes.

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Acknowledgements This study was supported by an educational grant from Schering Plough, Italy and by funds from MURST, Italy.

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