Journal of Antimicrobial Chemotherapy (1992) 29, 105-113
Purification and characterization of an imipenem hydrolysing metallo-fl-lactamase from Bacteroides fragitts M. Hedberg, C. Edhmd, L. Iindqvist, M. Rylander and C. E. Nord*
An imipenem resistant /Mactamase producing strain of Bacteroides fragilis was isolated from a clinical specimen. The specific activity of the unpurified /Uactamase was 5-5 U/mg protein. The /Mactamase was purified 60-fold by Q Sepharose, Sephacryl S-300 and Mono Q column passages. The strain was able to inactivate imipenem and cefoxitin in broth cultures. The enzyme hydrolysed imipenem more rapidly than ampicillin, benrylpcnicillin, cephalothin and cefoxitin. The activity of the enzyme was Zn2+ dependent and was completely inhibited by EDTA. The inhibition was reversed by ZnSO4. Preincubation with the common /Mactamase inhibitors clavulanic acid, sulbactam and tazobactam did not reduce the enzyme activity. The molecular weight was determined by sodium dodecyl sulfate gradient gel clectrophoresis to be 31,000 Daltons and the isoelectric point was 4-S.
Introduction
Members of the Bacteroides fragilis group are the most frequently isolated anaerobic pathogens from human infections. Clinical isolates are frequently resistant to penicillins and cephalosporins, while resistance to carbapenems, such as imipenem, is very rare. Different mechanisms are involved in /Mactam resistance: production of /Mactamases which inactivates the drug, alteration of penicillin-binding proteins which affects the affinity of the proteins for the /Mactam agent, and blocked penetration of the antibiotic through the bacterial outer membrane. The most important factor in /Mactam resistance in B. fragilis is the production of /Mactamases (Nord & Hedberg, 1990). The aim of the study was to purify and characterize a /Mactamase from a clinical human multiresistant isolate of B. fragilis. Materials and methods Bacterial strain
B. fragilis strain KSB1468/90 was isolated from the blood of a patient with septicaemia at the Karolinska Hospital, Stockholm, Sweden. The patient failed to respond to metronidazole therapy. The strain was identified according to Holdeman, Moore & Cato (1977). 'Corresponding author. 0305-7453/92/020105+09 $02.00/0
105 © 1992 The British Society for Antimicrobial Chemotherapy
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Department of Microbiology, Huddinge University Hospital, Huddinge, and Department of Clinical Microbiology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden
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Antimicrobial agents The following agents were tested: ampicillin, benzylpenicillin and clavulanic acid (Astra, Sodertalje, Sweden), ccfoxitin and imipenem (Merck Sharp & Dohme, Rahway, USA), cephalothin (Eli Lilly and Co., Indianapolis, USA), clindamycin (The Upjohn Company, Kalamazoo, USA), doxycycline and sulbactam (Pfizer, Griton, NY, USA), metronidazole (Rhone-Poulenc, Helsingborg, Sweden), nitrocefin (Glaxo Pharmaceuticals, Greenford, UK) and tazobactam (Lederle, Pearl River, USA). Antimicrobial susceptibility tests
Media The media used was brain heart infusion broth (Oxoid, Basingstoke, Hants, England) supplemented with 0-005% haemin and 0-5% yeast extracts (BHIS) according to Cuchural, Malamy & Tally (1986). Inactivation of imipenem and cefoxitin in broth cultures Overnight cultures of B.fragilis KSB1468/90 were used to inoculate BHIS-broth, containing 64 mg imipenem or cefoxitin per mL. As controls, B. fragilis ATCC 25285 and also broth with antimicrobial agents but without bacteria were used. All samples were incubated in duplicate with or without 0-1% Triton X-100 at 30°C for 0, 1, 4 and 24 h. After incubation the samples were centrifuged and the supernatant were assayed for residual imipenem and cefoxitin respectively by an agar diffusion assay using wells as diffusion centres. Production of fi-lactamase The B.fragilis KSB 1468/90 strain was cultured in a fermentor in a 3 L BHIS-broth (FL 103: BioTec, Stockholm, Sweden). Cultivation was performed at 37°C and pH 7-0 under N 2 atmosphere. The culture was harvested at maximum enzyme production in the late log phase (17 h) by centrifugation at 10,000 g for 30 min at 4°C. The cells were washed once in sodium phosphate buffer (0-01 M, pH 7-0) at 4°C and then disrupted by osmotic shock (Berg, 1981) and by 0-1% Triton X-100. The supernatant fractions were assayed for /Mactamase activity and used for further purification. Determination of fi-lactamase activity /J-Lactamase activity was assayed spectrophotometrically with nitrocefin (100 HM) in 5 mM sodium phosphate buffer (pH 7-0, 30°Q as the substrate (O'Callaghan et al., 1972). All determinations were carried out in duplicate. One unit of /Mactamase was defined as the amount which formed 1-0 /xmol of product per min under these conditions.
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The minimum inhibitory concentrations (MIC) were determined by the agar dilution method on PDM-agar (AB Biodisk, Solna, Sweden) with the addition of 5% defibrinated horse blood. The inoculum was approximately 10* cfu/mL, applied by a Steers replicator and incubated for 48 h at 37°C in anaerobic jars (GasPak, BBL Microbiologic Systems, Cockeysville, Md, USA).
hydroJysing fMactamase of B.
fragiSs
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Determination of protein The protein content was determined by the method of Lowry, Rosebrough & Randall (1951) with bovine serum albumin as the standard. Purification of fl-lactamase
SDS gradient gel electrophoresis and analytical isoelectric focusing The molecular weight and the isoelectric point were determined by Pharmacia Phast System, LKB, Stockholm, Sweden. The methods and standards used were as recommended by the manufacturer. Substrate profile Kn and Vnmx values of ampicillin, imipenem and nitrocefin were derived from reciprocal plots obtained by direct spectrophotometric assays. For benzylpenicillin, ampicillin, cefoxitin and cephalothin, K^ values were determined indirectly by competitive interaction between the antimicrobial agent and nitrocefin (Abraham & Waley, 1979; Kesado el al., 1989). The initial rate of hydrolysis of nitrocefin at seven different concentrations (10-300 HM) was determined in the presence of 002 U /Mactamase in a total volume of 3-0 mL. Each nitrocefin concentration was combined with five antimicrobial concentrations. The choice of antimicrobial concentrations were predetermined so that the apparent K^ for nitrocefin was increased by approximately factors two to eight. The apparent A,,, values for nitrocefin were plotted against the antibiotic concentrations to obtain the K^ for the antibiotic graphically.
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Step 1. The crude enzyme was dialysed against 20 mM Tris hydrochloride-buffer (pH 8-0) containing 20% glycerol and 0-1 mM ZnSO4 to a final volume of 800 mL. It was then passed through a Q Sepharose Fast Flow mass anion exchange column (2-0 x 40 cm; Pharmacia LKB Biotechnology AB, Uppsala, Sweden) equilibrated with 300 mL 20 HIM Tris hydrochloride (pH 80), 20% glycerol, 0-1 mM ZnSO4 and eluated by the same buffer with a gradient of 0 to 0-3 M NaCl. The flow rate was 2 mL/min. At 0-15-0-17 M NaCl, ten fractions (5 mL each) containing /Mactamase activity were collected and concentrated to 7-5 mL by ultrafiltration (Diaflo, PM 10; nominal molecular weight exclusion limit 10,000; Amicon Corporation, Scientific Systems Division, Danvers, MA, Ireland). Step 2. The concentrated /Mactamase sample from step 1 was passed through a S-300 Sephacryl gel filtration column (5-0 x 71 cm; Pharmacia) with 20 mM Tris hydrochloride (pH 8-0), 20% glycerol, 0-1 mM ZnSO4 as the eluent. The effluent was collected in 5-0 mL fractions and the flow rate was 0-5 mL/min. Five fractions, no. 17-21, containing /Mactamase activity were pooled. Step 3. The pooled fractions were further purified by anion-exchange chromatography, Mono Q (HR 5/5; fast protein liquid chromatography [FPLC] system; Pharmacia) equilibrated with 20 mM Tris hydrochloride (pH 8-0), 20% glycerol, 0-1 mM ZnSO4. The sample was processed in two consecutive runs. The chromatography was developed with 20 mM Tris hydrochloride (pH 80), 20% glycerol, 0-1 mM ZnSO4 containing a gradient (25 mL) of 0-0-3 M NaCl. The effluent was collected in 0-5 mL fractions and the flow rate was 0-6 mL/min.
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V^ values for the different antibiotics were calculated from the Henri Michaelis Menten equation by using the K^ values obtained by the indirect determination and the initial rates obtained from the hydrolysis of benzylpenicillin and ampicillin at final concentrations of 500 /IM and cephalothin and cefoxitih at 100 /*M. The initial rates were obtained by a direct spectrophotometric method based on the difference in light absorption between substrate and product at a specified wavelength (Waley, 1974; Samuni, 1975). The relative maximal hydrolysis rate was obtained by setting V^ for benzylpenicillin at 100. Inhibition studies
Results Antimicrobial susceptibility pattern B.fragilis strain KSB1468/90 was highly resistant to benzylpenicillin and ampicillin (MIC > 128 mg/L), imipenem (MIC = 128 mg/L) and cefoxitin (MIC = 64 mg/L), moderate resistant to doxycycline (MIC = 4 mg/L) and metronidazole (MIC = 16 mg/L) and susceptible to clindamycin (MIC = 0-5 mg/L) and chloramphenicol (MIC = 1 mg/L). Inactivation of imipenem and cefoxitin B.fragilis strain KSB 1468/90 was able to inactivate both imipenem and cefoxitin in broth cultures. After 1 h incubation with imipenem and after 4 h incubation with cefoxitin, in the presence of 0-1% Triton X-100, less than 0-5% of the original antibiotic remained compared to incubation with the control strain under the same conditions. fl-Lactamase formation and purification Qualitative detection of /Mactamase showed that B.fragilis KSB 1468/90 was a high producer of /Mactamase. The enzyme activity was only found intracellularly. The specific activity of /Mactamase in the supernatant of disrupted cells was 5-5 U/mg protein. The results of each purification step are presented in Table I. In the first Q Sepharose Fast Flow mass anion exchange column was used to concentrate the enzyme and to remove large amounts of contaminating^proteins. In step 2 Sephacryl S-300 chromatography was used to separate the enzyme from proteins of other molecular sizes and to change the solvent to a more useful one for the next step.
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Clavulanic acid, sulbactam and tazobactam in various concentrations (0-1, 10 and 300 DIM) were preincubated for 0-5, 20 and 90 min at 30cC with 0-1 U/mL of the purified /Mactamase to study the inhibition of the enzyme activity. The remaining activity was determined by the nitrocefin assay. As controls, 3-0 fM of the different /Mactamase inhibitors were preincubated in the same way together with a /Mactamase (0-1 U/mL) from a strain of Bacteroides uniformis. The B. vniformis enzyme activity is shown to be reduced by these three inhibitors (Hedberg, M., Lindqvist, L. Tuner, K. & Nord, C. E., unpublished data). Per cent inhibition was calculated at 100 x [(c-r)/c], where c is the activity in control samples incubated without inhibitor and r is the remaining activity in samples incubated with inhibitor. The activity of the /Mactamase was also tested in the presence of 1 mM EDTA.
Tmlpfreni bydrotysiiig fl-lactamase of B. fragiEs
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Table L Purification of /J-lactamase from B.fragUis strain KSB 1468/90
Stq)
Spec, activity (U/mg of protein)
530 320
5-5 51
250 60
Purification (fold)
Recovery (%)
9-3
604
63
11-5
47-2
320
58-2
11-3
Finally, the enzyme was purified about 60-fold by two cycles of anion-exchange chromatography on Mono Q columns by FPLC. This step resulted in a single peak of 0-lactamase (Figure 1). The protein yield was 0-185 mg protein per mL. The purified enzyme was stable in 20% glycerol and in the presence of 0-1 mi* ZnJ+-ions. It was stored at +4°C. The purified enzyme without addition of Zn2+-ions was stable at -20°C but unstable at +4°C. Molecular weight and isoelectric point
The molecular weight of the purified /Mactamase was determined to be 31,000 Daltons, and the isoelectric point was estimated to be 4-5.
60 080 SO
C-15
4O
S 30 0-10 20 C-O5
10
OOO' •
^
•
•*•!
20 25 Fraction no.
? • • 35 '•
•
*0
40
Flgm 1. Last ftep in purification of /Mactamase from B.fragUis strain KSB 1468/90 by anion-exchange chromatography Mono Q (HR 5/5; FPLC). D, Ab*orbance at 280 nnj; • , U/mL.
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Crude extract (1) Mass anion exchange on Q Sepharose fast flow (2) Gel filtration on Sephacryl S-300 (3) Mass anion exchange on Mono Q column
Total activity
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Tabk IL Kinetic parameters of the purified 0-lactamase from B. fragilis strain KSB 1468/90 /J-lactam agent (substrate) 20-4 125 133 50 284 380 6-9
Benzylpenicillin' Ampicillin' Ampicillin Cephalothin* Cefoxitin* Imipenem Nitrocefin
VnJKm
/xmol/min per mg
relative
relative
109 136 130 37-7 101 322 153
100 120 119 359-2 290 140
100 20 18 140 065 16 400
Substrate profile The enzyme was able to hydrolyse both penicillins and cephalosporins as well as imipenem. The kinetic parameters for the various /f-lactam antibiotics hydrolysed by the purified /Mactamase are shown in Table II. Inhibition studies The enzyme activity was completely inhibited by 1 mix EDTA, and the activity of a two-fold dilution of the EDTA-treated enzyme was completely reversed by 1 HIM 100
80
60
20
20
40
60
BO
100
Tient (mln)
Figure 2. Effect of three /Hactamaje inhibitor* on the /Mactamase activity. A . Oavulanic acid; • , sulbactam; • , tazobactam. As a control, 3-0 /JM of the different /J-lactamaje inhibitors were preincubated at 30°C together with a /Mactamase (0-1 U/mL) from a positive control strain of B. untformis. A . Clavulank acid; D , lulbactam; O. tazobactam.
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'Kn values obtained from indirect spectrophotometric assays.
Imipenem hydrotyring 0-lKtamase of B.
fragilis
HI
ZnSO4. The /Mactamase activity was not inhibited by clavulanic acid, sulbactam or tazobactam after preincubation during 90 min with 300 /IM inhibitor (Figure 2). Discussion
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The frequency of /Mactamase production by various Bacteroides species is high. Seventy-five—100% of strains in the B.fragilis group are reported to be /Mactamase producers (Finegold, 1989). /}-Lactamases of anaerobic bacteria especially B.fragilis have been extensively investigated during recent years (Nord & Hedberg, 1990). The enzymes produced by the B.fragilis group usually hydrolyse cephalosporins more rapidly than penicillins while they have a poor activity against cephamycins and carbapenems. To determine the K^ values for the /Mactam antibiotics, the indirect method was used, i.e. inhibition of the nitrocefin hydrolysis by the /Mactam antibiotics. This method is suitable when large ranges of /Mactam concentrations are investigated. In the indirect assay, the /Mactam antibiotic is hydrolysed concomitantly with nitrocefin. However, the influence of the decreased /Mactam concentration on the nitrocefin hydrolysis rate can be controlled by the relation of the Vm values for nitrocefin and the /Mactam antibiotic. If Vmix for the /Mactam antibiotic is lower or in the same order as Vma for nitrocefin, the /Mactam hydrolysis does not significantly influence the initial rate of nitrocefin hydrolysis. Also, if the /Mactam antibiotic is hydrolysed much faster than nitrocefin, the rate of nitrocefin hydrolysis will increase during the observation period and does not give a linear absorbance recording versus time. The /Mactamase characterized in the present study hydrolysed imipenem more rapidly than all the other /Mactam agents studied. The Michaelis constant K^ is an equilibrium constant equal to the substrate concentration that will be hydrolysed at a rate equal to 0-5 Vatx. Despite therelativehigh K^ for imipenem (380 /XM = 120 mg/L), the hydrolysing rate of imipenem will be approximately 0-5 V^ = 160 /anol/min per mg enzyme or 50 mg/min per mg enzyme at the MIC (128 mg/L), which is considerably high. This is in accordance with the result that imipenem was shown to be very quickly inactivated in broth cultures. The lower maximum hydrolysing rate for cefoxitin corresponds to the slower inactivation of the drug in broth cultures. Resistance to cefoxitin has previously been associated with decreased penetration of the drug through the bacterial outer membrane (Piddock & Wise, 1987). Imipenem hydrolysing /Mactamases with Zn2+ as a co-factor produced by Bacillus cereus, Pseudomonas maltophilia and Flavobacterium odoratum have been described earlier (Sato et a!., 1985; Bush, 1989). These enzymes belong to group 3 according to the classification of /Mactamases by Bush (1989). Enzymes in this group require a metal ion for enzymatic activity and are often not inhibited by clavulanic acid. There have been a few reports on extended /Mactamase activity in B. fragilis. Yotsuji et al. (1983) reported a broad spectrum /Mactamase produced by B.fragilis with a substrate profile similar to the enzyme reported in the present study. However, their isolate was susceptible to imipenem in vitro (MIC =12-5 mg/L) and they did not report about EDTA inhibition or effect of metal ions. Cuchural et al. (1986) also described a broad spectrum /Mactamase from two strains of B. fragilis hydrolysing both cefoxitin and imipenem. The activity was completely inhibited by EDTA and had Zn2+ as a co-factor. The activity was similar to the B. fragilis KSB1468/90 enzyme not inhibited
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References Abraham, E. P. & Waley, S. G. (1979). /?-Lactamases from Bacillus cereus. In Beta-lactamases (Hamilton-Miller, J. M. T. & Smith, J. T., Eds), pp. 311-38. Academic Press, London. Bandoh, K.F Muto, Y., Watanabe, K... Katoh, N. & Ucno, K. (1991). Biochemical properties and purification of metallo-/J-lactamase from Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 35, 371-2. Berg, J.-O. (1981). Cellular localization of glycoside hydrolases in Bacteroides fragilis. Current Microbiology 5, 13-7. Bush, K. (1989). Characterization of /Mactamases. Antimicrobial Agents and Chemotherapy 33, 259-63. Cuchural, G. J., Malamy, M. H. & Tally, F. P. (1986). 0-Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 30, 645-8. Finegold, S. M. (1989). Mechanisms of resistance in anaerobes and new developments in testing. Diagnostic Microbiology and Infectious Diseases 12, 4 Suppl., 117S-20S. Holdeman, L. V., Cato, E. P. & Moore, E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute and State University, Blacksburg, VA. Kesado, T., Lindqvist, L., Hedberg, M., Tuner, K. & Nord, C. E. (1989). Purification and characterization of a new /Mactamase from Clostridium butyricum. Antimicrobial Agents and Chemotherapy 33, 1302-7. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent Journal of Biological Chemistry 193, 265-75. Nord, C. E. & Hedberg, M. (1990). Resistance to /Mactam antibiotics in anaerobic bacteria. Reviews of Infectious Diseases 12, Suppl. 2, S231-4.
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by the /Mactamase inhibitors clavulanic acid or sulbactam. The molecular weights were estimated to be 44,000 Daltons, and the isoelectric point was not determined. A recent study by Thompson & Malamy (1990) has reported that the molecular weight of this carbapenemase is now calculated to be 27,260. In contrast to the KSB1468/90 enzyme, freezing of the /Mactamases resulted in > 99% loss of activity. The authors suggested that decreased outer membrane permeability and /Mactamase activity synergistically contributed to the /Mactam resistance. Recently, another broad-spectrum metallo /Mactamase from an imipenem resistant B. fragilis strain was reported by Bandoh et al. (1991). The substrate profile of this enzyme differed from that of our enzyme, while other characteristics, like inhibition profiles and physical properties, were similar to those found in the present study. Despite some discrepancies in the properties of the new imipenem-hydrolysing /Mactamases from B. fragilis reported so far, it seems that a new class of enzymes produced by anaerobic bacteria have been recognized. Although resistance to imipenem and cefoxitin among B. fragilis seems to be very rare, the discovery of these new broad range /Mactamases is clinically important These strains are all clinical isolates recovered from human infections. The fact that these enzymes are not inhibited by common /Mactamase inhibitors is also a cause of concern. Serious infections caused by /Mactamase producing B. fragilis strains are commonly treated with cefoxitin or imipenem. Increased use of these /Mactamase stable compounds may result in new strategies by the micro-organisms to survive. The B. fragilis strain described in the present study was also resistant to metronidazole which reduces further the available therapeutic range of drugs. Multiresistant B. fragilis strains will probably be an increasing clinical problem in the future.
hvdrorysing fMsctamase of B. fragilis
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{Received 24 June 1991; revised version accepted 22 October 1991)
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O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method fendetection of 0-lactamases by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 283-8. Piddock, L. J. V. & Wise, R. (1987). Cefoxitin resistance in Bacteroides species: evidence indicating two mechanisms causing decreased susceptibility. Journal of Antimicrobial Chemotherapy 19, 161-70. Samuni, A. (1975). A direct spectrophotometric assay and determination of Michaelis constants for the /Mactamase reaction. Analytical Biochemistry 63, 17-26. Sato, K., Fujii, T., Okamoto, R., Inoue, M. & Mitsuhashi, S. (1985). Biochemical properties of /Mactamase produced by Flavobacterium odora turn. Antimicrobial Agents and Chemotherapy 27, 612-4. Thompson, S. & Malamy, M. H. (1990). Sequencing the gene for an imipenem-cefoxitinhydrolysing enzyme (CfiA) from Bacteroides fragilis TAL 2480 reveals strong similarity between CfiA and Bacillus cereus 0-lactamase EL Journal of Bacteriology 172, 2584-93. Waley, S. G. (1974). A spectrophotometric assay of /J-lactamase action on penicillins. Biochemical Journal 139, 789-90. Yotsuji, A., Minami, S., Inoue, M. & Mitsuhashi, S. (1983). Properties of novel ^-lactamase produced by Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 24, 925-9.
Purification and characterization of an imipenem hydrolysing metallo-fl-lactamase from Bacteroides fragitts M. Hedberg, C. Edhmd, L. Iindqvist, M. Rylander and C. E. Nord*
An imipenem resistant /Mactamase producing strain of Bacteroides fragilis was isolated from a clinical specimen. The specific activity of the unpurified /Uactamase was 5-5 U/mg protein. The /Mactamase was purified 60-fold by Q Sepharose, Sephacryl S-300 and Mono Q column passages. The strain was able to inactivate imipenem and cefoxitin in broth cultures. The enzyme hydrolysed imipenem more rapidly than ampicillin, benrylpcnicillin, cephalothin and cefoxitin. The activity of the enzyme was Zn2+ dependent and was completely inhibited by EDTA. The inhibition was reversed by ZnSO4. Preincubation with the common /Mactamase inhibitors clavulanic acid, sulbactam and tazobactam did not reduce the enzyme activity. The molecular weight was determined by sodium dodecyl sulfate gradient gel clectrophoresis to be 31,000 Daltons and the isoelectric point was 4-S.
Introduction
Members of the Bacteroides fragilis group are the most frequently isolated anaerobic pathogens from human infections. Clinical isolates are frequently resistant to penicillins and cephalosporins, while resistance to carbapenems, such as imipenem, is very rare. Different mechanisms are involved in /Mactam resistance: production of /Mactamases which inactivates the drug, alteration of penicillin-binding proteins which affects the affinity of the proteins for the /Mactam agent, and blocked penetration of the antibiotic through the bacterial outer membrane. The most important factor in /Mactam resistance in B. fragilis is the production of /Mactamases (Nord & Hedberg, 1990). The aim of the study was to purify and characterize a /Mactamase from a clinical human multiresistant isolate of B. fragilis. Materials and methods Bacterial strain
B. fragilis strain KSB1468/90 was isolated from the blood of a patient with septicaemia at the Karolinska Hospital, Stockholm, Sweden. The patient failed to respond to metronidazole therapy. The strain was identified according to Holdeman, Moore & Cato (1977). 'Corresponding author. 0305-7453/92/020105+09 $02.00/0
105 © 1992 The British Society for Antimicrobial Chemotherapy
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Department of Microbiology, Huddinge University Hospital, Huddinge, and Department of Clinical Microbiology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden
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Antimicrobial agents The following agents were tested: ampicillin, benzylpenicillin and clavulanic acid (Astra, Sodertalje, Sweden), ccfoxitin and imipenem (Merck Sharp & Dohme, Rahway, USA), cephalothin (Eli Lilly and Co., Indianapolis, USA), clindamycin (The Upjohn Company, Kalamazoo, USA), doxycycline and sulbactam (Pfizer, Griton, NY, USA), metronidazole (Rhone-Poulenc, Helsingborg, Sweden), nitrocefin (Glaxo Pharmaceuticals, Greenford, UK) and tazobactam (Lederle, Pearl River, USA). Antimicrobial susceptibility tests
Media The media used was brain heart infusion broth (Oxoid, Basingstoke, Hants, England) supplemented with 0-005% haemin and 0-5% yeast extracts (BHIS) according to Cuchural, Malamy & Tally (1986). Inactivation of imipenem and cefoxitin in broth cultures Overnight cultures of B.fragilis KSB1468/90 were used to inoculate BHIS-broth, containing 64 mg imipenem or cefoxitin per mL. As controls, B. fragilis ATCC 25285 and also broth with antimicrobial agents but without bacteria were used. All samples were incubated in duplicate with or without 0-1% Triton X-100 at 30°C for 0, 1, 4 and 24 h. After incubation the samples were centrifuged and the supernatant were assayed for residual imipenem and cefoxitin respectively by an agar diffusion assay using wells as diffusion centres. Production of fi-lactamase The B.fragilis KSB 1468/90 strain was cultured in a fermentor in a 3 L BHIS-broth (FL 103: BioTec, Stockholm, Sweden). Cultivation was performed at 37°C and pH 7-0 under N 2 atmosphere. The culture was harvested at maximum enzyme production in the late log phase (17 h) by centrifugation at 10,000 g for 30 min at 4°C. The cells were washed once in sodium phosphate buffer (0-01 M, pH 7-0) at 4°C and then disrupted by osmotic shock (Berg, 1981) and by 0-1% Triton X-100. The supernatant fractions were assayed for /Mactamase activity and used for further purification. Determination of fi-lactamase activity /J-Lactamase activity was assayed spectrophotometrically with nitrocefin (100 HM) in 5 mM sodium phosphate buffer (pH 7-0, 30°Q as the substrate (O'Callaghan et al., 1972). All determinations were carried out in duplicate. One unit of /Mactamase was defined as the amount which formed 1-0 /xmol of product per min under these conditions.
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The minimum inhibitory concentrations (MIC) were determined by the agar dilution method on PDM-agar (AB Biodisk, Solna, Sweden) with the addition of 5% defibrinated horse blood. The inoculum was approximately 10* cfu/mL, applied by a Steers replicator and incubated for 48 h at 37°C in anaerobic jars (GasPak, BBL Microbiologic Systems, Cockeysville, Md, USA).
hydroJysing fMactamase of B.
fragiSs
107
Determination of protein The protein content was determined by the method of Lowry, Rosebrough & Randall (1951) with bovine serum albumin as the standard. Purification of fl-lactamase
SDS gradient gel electrophoresis and analytical isoelectric focusing The molecular weight and the isoelectric point were determined by Pharmacia Phast System, LKB, Stockholm, Sweden. The methods and standards used were as recommended by the manufacturer. Substrate profile Kn and Vnmx values of ampicillin, imipenem and nitrocefin were derived from reciprocal plots obtained by direct spectrophotometric assays. For benzylpenicillin, ampicillin, cefoxitin and cephalothin, K^ values were determined indirectly by competitive interaction between the antimicrobial agent and nitrocefin (Abraham & Waley, 1979; Kesado el al., 1989). The initial rate of hydrolysis of nitrocefin at seven different concentrations (10-300 HM) was determined in the presence of 002 U /Mactamase in a total volume of 3-0 mL. Each nitrocefin concentration was combined with five antimicrobial concentrations. The choice of antimicrobial concentrations were predetermined so that the apparent K^ for nitrocefin was increased by approximately factors two to eight. The apparent A,,, values for nitrocefin were plotted against the antibiotic concentrations to obtain the K^ for the antibiotic graphically.
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Step 1. The crude enzyme was dialysed against 20 mM Tris hydrochloride-buffer (pH 8-0) containing 20% glycerol and 0-1 mM ZnSO4 to a final volume of 800 mL. It was then passed through a Q Sepharose Fast Flow mass anion exchange column (2-0 x 40 cm; Pharmacia LKB Biotechnology AB, Uppsala, Sweden) equilibrated with 300 mL 20 HIM Tris hydrochloride (pH 80), 20% glycerol, 0-1 mM ZnSO4 and eluated by the same buffer with a gradient of 0 to 0-3 M NaCl. The flow rate was 2 mL/min. At 0-15-0-17 M NaCl, ten fractions (5 mL each) containing /Mactamase activity were collected and concentrated to 7-5 mL by ultrafiltration (Diaflo, PM 10; nominal molecular weight exclusion limit 10,000; Amicon Corporation, Scientific Systems Division, Danvers, MA, Ireland). Step 2. The concentrated /Mactamase sample from step 1 was passed through a S-300 Sephacryl gel filtration column (5-0 x 71 cm; Pharmacia) with 20 mM Tris hydrochloride (pH 8-0), 20% glycerol, 0-1 mM ZnSO4 as the eluent. The effluent was collected in 5-0 mL fractions and the flow rate was 0-5 mL/min. Five fractions, no. 17-21, containing /Mactamase activity were pooled. Step 3. The pooled fractions were further purified by anion-exchange chromatography, Mono Q (HR 5/5; fast protein liquid chromatography [FPLC] system; Pharmacia) equilibrated with 20 mM Tris hydrochloride (pH 8-0), 20% glycerol, 0-1 mM ZnSO4. The sample was processed in two consecutive runs. The chromatography was developed with 20 mM Tris hydrochloride (pH 80), 20% glycerol, 0-1 mM ZnSO4 containing a gradient (25 mL) of 0-0-3 M NaCl. The effluent was collected in 0-5 mL fractions and the flow rate was 0-6 mL/min.
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V^ values for the different antibiotics were calculated from the Henri Michaelis Menten equation by using the K^ values obtained by the indirect determination and the initial rates obtained from the hydrolysis of benzylpenicillin and ampicillin at final concentrations of 500 /IM and cephalothin and cefoxitih at 100 /*M. The initial rates were obtained by a direct spectrophotometric method based on the difference in light absorption between substrate and product at a specified wavelength (Waley, 1974; Samuni, 1975). The relative maximal hydrolysis rate was obtained by setting V^ for benzylpenicillin at 100. Inhibition studies
Results Antimicrobial susceptibility pattern B.fragilis strain KSB1468/90 was highly resistant to benzylpenicillin and ampicillin (MIC > 128 mg/L), imipenem (MIC = 128 mg/L) and cefoxitin (MIC = 64 mg/L), moderate resistant to doxycycline (MIC = 4 mg/L) and metronidazole (MIC = 16 mg/L) and susceptible to clindamycin (MIC = 0-5 mg/L) and chloramphenicol (MIC = 1 mg/L). Inactivation of imipenem and cefoxitin B.fragilis strain KSB 1468/90 was able to inactivate both imipenem and cefoxitin in broth cultures. After 1 h incubation with imipenem and after 4 h incubation with cefoxitin, in the presence of 0-1% Triton X-100, less than 0-5% of the original antibiotic remained compared to incubation with the control strain under the same conditions. fl-Lactamase formation and purification Qualitative detection of /Mactamase showed that B.fragilis KSB 1468/90 was a high producer of /Mactamase. The enzyme activity was only found intracellularly. The specific activity of /Mactamase in the supernatant of disrupted cells was 5-5 U/mg protein. The results of each purification step are presented in Table I. In the first Q Sepharose Fast Flow mass anion exchange column was used to concentrate the enzyme and to remove large amounts of contaminating^proteins. In step 2 Sephacryl S-300 chromatography was used to separate the enzyme from proteins of other molecular sizes and to change the solvent to a more useful one for the next step.
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Clavulanic acid, sulbactam and tazobactam in various concentrations (0-1, 10 and 300 DIM) were preincubated for 0-5, 20 and 90 min at 30cC with 0-1 U/mL of the purified /Mactamase to study the inhibition of the enzyme activity. The remaining activity was determined by the nitrocefin assay. As controls, 3-0 fM of the different /Mactamase inhibitors were preincubated in the same way together with a /Mactamase (0-1 U/mL) from a strain of Bacteroides uniformis. The B. vniformis enzyme activity is shown to be reduced by these three inhibitors (Hedberg, M., Lindqvist, L. Tuner, K. & Nord, C. E., unpublished data). Per cent inhibition was calculated at 100 x [(c-r)/c], where c is the activity in control samples incubated without inhibitor and r is the remaining activity in samples incubated with inhibitor. The activity of the /Mactamase was also tested in the presence of 1 mM EDTA.
Tmlpfreni bydrotysiiig fl-lactamase of B. fragiEs
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Table L Purification of /J-lactamase from B.fragUis strain KSB 1468/90
Stq)
Spec, activity (U/mg of protein)
530 320
5-5 51
250 60
Purification (fold)
Recovery (%)
9-3
604
63
11-5
47-2
320
58-2
11-3
Finally, the enzyme was purified about 60-fold by two cycles of anion-exchange chromatography on Mono Q columns by FPLC. This step resulted in a single peak of 0-lactamase (Figure 1). The protein yield was 0-185 mg protein per mL. The purified enzyme was stable in 20% glycerol and in the presence of 0-1 mi* ZnJ+-ions. It was stored at +4°C. The purified enzyme without addition of Zn2+-ions was stable at -20°C but unstable at +4°C. Molecular weight and isoelectric point
The molecular weight of the purified /Mactamase was determined to be 31,000 Daltons, and the isoelectric point was estimated to be 4-5.
60 080 SO
C-15
4O
S 30 0-10 20 C-O5
10
OOO' •
^
•
•*•!
20 25 Fraction no.
? • • 35 '•
•
*0
40
Flgm 1. Last ftep in purification of /Mactamase from B.fragUis strain KSB 1468/90 by anion-exchange chromatography Mono Q (HR 5/5; FPLC). D, Ab*orbance at 280 nnj; • , U/mL.
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Crude extract (1) Mass anion exchange on Q Sepharose fast flow (2) Gel filtration on Sephacryl S-300 (3) Mass anion exchange on Mono Q column
Total activity
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M Hedberg et aL
Tabk IL Kinetic parameters of the purified 0-lactamase from B. fragilis strain KSB 1468/90 /J-lactam agent (substrate) 20-4 125 133 50 284 380 6-9
Benzylpenicillin' Ampicillin' Ampicillin Cephalothin* Cefoxitin* Imipenem Nitrocefin
VnJKm
/xmol/min per mg
relative
relative
109 136 130 37-7 101 322 153
100 120 119 359-2 290 140
100 20 18 140 065 16 400
Substrate profile The enzyme was able to hydrolyse both penicillins and cephalosporins as well as imipenem. The kinetic parameters for the various /f-lactam antibiotics hydrolysed by the purified /Mactamase are shown in Table II. Inhibition studies The enzyme activity was completely inhibited by 1 mix EDTA, and the activity of a two-fold dilution of the EDTA-treated enzyme was completely reversed by 1 HIM 100
80
60
20
20
40
60
BO
100
Tient (mln)
Figure 2. Effect of three /Hactamaje inhibitor* on the /Mactamase activity. A . Oavulanic acid; • , sulbactam; • , tazobactam. As a control, 3-0 /JM of the different /J-lactamaje inhibitors were preincubated at 30°C together with a /Mactamase (0-1 U/mL) from a positive control strain of B. untformis. A . Clavulank acid; D , lulbactam; O. tazobactam.
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'Kn values obtained from indirect spectrophotometric assays.
Imipenem hydrotyring 0-lKtamase of B.
fragilis
HI
ZnSO4. The /Mactamase activity was not inhibited by clavulanic acid, sulbactam or tazobactam after preincubation during 90 min with 300 /IM inhibitor (Figure 2). Discussion
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The frequency of /Mactamase production by various Bacteroides species is high. Seventy-five—100% of strains in the B.fragilis group are reported to be /Mactamase producers (Finegold, 1989). /}-Lactamases of anaerobic bacteria especially B.fragilis have been extensively investigated during recent years (Nord & Hedberg, 1990). The enzymes produced by the B.fragilis group usually hydrolyse cephalosporins more rapidly than penicillins while they have a poor activity against cephamycins and carbapenems. To determine the K^ values for the /Mactam antibiotics, the indirect method was used, i.e. inhibition of the nitrocefin hydrolysis by the /Mactam antibiotics. This method is suitable when large ranges of /Mactam concentrations are investigated. In the indirect assay, the /Mactam antibiotic is hydrolysed concomitantly with nitrocefin. However, the influence of the decreased /Mactam concentration on the nitrocefin hydrolysis rate can be controlled by the relation of the Vm values for nitrocefin and the /Mactam antibiotic. If Vmix for the /Mactam antibiotic is lower or in the same order as Vma for nitrocefin, the /Mactam hydrolysis does not significantly influence the initial rate of nitrocefin hydrolysis. Also, if the /Mactam antibiotic is hydrolysed much faster than nitrocefin, the rate of nitrocefin hydrolysis will increase during the observation period and does not give a linear absorbance recording versus time. The /Mactamase characterized in the present study hydrolysed imipenem more rapidly than all the other /Mactam agents studied. The Michaelis constant K^ is an equilibrium constant equal to the substrate concentration that will be hydrolysed at a rate equal to 0-5 Vatx. Despite therelativehigh K^ for imipenem (380 /XM = 120 mg/L), the hydrolysing rate of imipenem will be approximately 0-5 V^ = 160 /anol/min per mg enzyme or 50 mg/min per mg enzyme at the MIC (128 mg/L), which is considerably high. This is in accordance with the result that imipenem was shown to be very quickly inactivated in broth cultures. The lower maximum hydrolysing rate for cefoxitin corresponds to the slower inactivation of the drug in broth cultures. Resistance to cefoxitin has previously been associated with decreased penetration of the drug through the bacterial outer membrane (Piddock & Wise, 1987). Imipenem hydrolysing /Mactamases with Zn2+ as a co-factor produced by Bacillus cereus, Pseudomonas maltophilia and Flavobacterium odoratum have been described earlier (Sato et a!., 1985; Bush, 1989). These enzymes belong to group 3 according to the classification of /Mactamases by Bush (1989). Enzymes in this group require a metal ion for enzymatic activity and are often not inhibited by clavulanic acid. There have been a few reports on extended /Mactamase activity in B. fragilis. Yotsuji et al. (1983) reported a broad spectrum /Mactamase produced by B.fragilis with a substrate profile similar to the enzyme reported in the present study. However, their isolate was susceptible to imipenem in vitro (MIC =12-5 mg/L) and they did not report about EDTA inhibition or effect of metal ions. Cuchural et al. (1986) also described a broad spectrum /Mactamase from two strains of B. fragilis hydrolysing both cefoxitin and imipenem. The activity was completely inhibited by EDTA and had Zn2+ as a co-factor. The activity was similar to the B. fragilis KSB1468/90 enzyme not inhibited
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References Abraham, E. P. & Waley, S. G. (1979). /?-Lactamases from Bacillus cereus. In Beta-lactamases (Hamilton-Miller, J. M. T. & Smith, J. T., Eds), pp. 311-38. Academic Press, London. Bandoh, K.F Muto, Y., Watanabe, K... Katoh, N. & Ucno, K. (1991). Biochemical properties and purification of metallo-/J-lactamase from Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 35, 371-2. Berg, J.-O. (1981). Cellular localization of glycoside hydrolases in Bacteroides fragilis. Current Microbiology 5, 13-7. Bush, K. (1989). Characterization of /Mactamases. Antimicrobial Agents and Chemotherapy 33, 259-63. Cuchural, G. J., Malamy, M. H. & Tally, F. P. (1986). 0-Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 30, 645-8. Finegold, S. M. (1989). Mechanisms of resistance in anaerobes and new developments in testing. Diagnostic Microbiology and Infectious Diseases 12, 4 Suppl., 117S-20S. Holdeman, L. V., Cato, E. P. & Moore, E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute and State University, Blacksburg, VA. Kesado, T., Lindqvist, L., Hedberg, M., Tuner, K. & Nord, C. E. (1989). Purification and characterization of a new /Mactamase from Clostridium butyricum. Antimicrobial Agents and Chemotherapy 33, 1302-7. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent Journal of Biological Chemistry 193, 265-75. Nord, C. E. & Hedberg, M. (1990). Resistance to /Mactam antibiotics in anaerobic bacteria. Reviews of Infectious Diseases 12, Suppl. 2, S231-4.
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by the /Mactamase inhibitors clavulanic acid or sulbactam. The molecular weights were estimated to be 44,000 Daltons, and the isoelectric point was not determined. A recent study by Thompson & Malamy (1990) has reported that the molecular weight of this carbapenemase is now calculated to be 27,260. In contrast to the KSB1468/90 enzyme, freezing of the /Mactamases resulted in > 99% loss of activity. The authors suggested that decreased outer membrane permeability and /Mactamase activity synergistically contributed to the /Mactam resistance. Recently, another broad-spectrum metallo /Mactamase from an imipenem resistant B. fragilis strain was reported by Bandoh et al. (1991). The substrate profile of this enzyme differed from that of our enzyme, while other characteristics, like inhibition profiles and physical properties, were similar to those found in the present study. Despite some discrepancies in the properties of the new imipenem-hydrolysing /Mactamases from B. fragilis reported so far, it seems that a new class of enzymes produced by anaerobic bacteria have been recognized. Although resistance to imipenem and cefoxitin among B. fragilis seems to be very rare, the discovery of these new broad range /Mactamases is clinically important These strains are all clinical isolates recovered from human infections. The fact that these enzymes are not inhibited by common /Mactamase inhibitors is also a cause of concern. Serious infections caused by /Mactamase producing B. fragilis strains are commonly treated with cefoxitin or imipenem. Increased use of these /Mactamase stable compounds may result in new strategies by the micro-organisms to survive. The B. fragilis strain described in the present study was also resistant to metronidazole which reduces further the available therapeutic range of drugs. Multiresistant B. fragilis strains will probably be an increasing clinical problem in the future.
hvdrorysing fMsctamase of B. fragilis
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{Received 24 June 1991; revised version accepted 22 October 1991)
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O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method fendetection of 0-lactamases by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 283-8. Piddock, L. J. V. & Wise, R. (1987). Cefoxitin resistance in Bacteroides species: evidence indicating two mechanisms causing decreased susceptibility. Journal of Antimicrobial Chemotherapy 19, 161-70. Samuni, A. (1975). A direct spectrophotometric assay and determination of Michaelis constants for the /Mactamase reaction. Analytical Biochemistry 63, 17-26. Sato, K., Fujii, T., Okamoto, R., Inoue, M. & Mitsuhashi, S. (1985). Biochemical properties of /Mactamase produced by Flavobacterium odora turn. Antimicrobial Agents and Chemotherapy 27, 612-4. Thompson, S. & Malamy, M. H. (1990). Sequencing the gene for an imipenem-cefoxitinhydrolysing enzyme (CfiA) from Bacteroides fragilis TAL 2480 reveals strong similarity between CfiA and Bacillus cereus 0-lactamase EL Journal of Bacteriology 172, 2584-93. Waley, S. G. (1974). A spectrophotometric assay of /J-lactamase action on penicillins. Biochemical Journal 139, 789-90. Yotsuji, A., Minami, S., Inoue, M. & Mitsuhashi, S. (1983). Properties of novel ^-lactamase produced by Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 24, 925-9.
