a Antibiotic Reference Unit, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK b Catedra de Infectologia y Microbiologia Clinica, Facultad de Post Grado en Ciencias de la Salud, Universidad Catolica Argentina, Adolfo Davila 1500, 1107 Buenos Aires, Argentina
Abstract
Three carbapenem-resistant Acinetobacter baumannii isolates were collected at a hospital in Buenos Aires, Argentina. Isoelectric focusing revealed multiple ß-lactamases, with two of the isolates showing identical profiles. A pI 6.9 carbapenemase with a molecular weight of 30 kDa was purified from one of these two isolates. The enzyme was predominantly a penicillinase, with its highest Vmax for oxacillin but highest VmaxKmfor benzylpenicillin. First-generation cephalosporins and imipenem were weaker substrates than penicillins, and oxyimino-aminothiazolyl cephalosporins were essentially stable. Meropenem-hydrolysing activity was not detected, despite resistance. The carbapenemase was inhibited by clavulanic acid and tazobactam, but not by EDTA. These kinetics place the enzyme into functional group 2; as an oxacillinase it could be placed in sub-group 2d or, as a zinc-independent carbapenemase, in sub-group 2f.
Introduction
Acinetobacter baumannii is a frequent opportunistic pathogen and is often resistant to multiple antibiotics. 1 ,2 Carbapenems have retained better activity than other agents, but reports of resistance are now accumulating. A carbapenem-resistant A. baumannii isolate was found from Scotland in 1985 3 and produced ARI-1, a weak carbapenemase. Carbapenemase-producing acinetobacter isolates have since been reported from Argentina, 4 ,5 Belgium, 5 Brazil, 6 Cuba, 7 England,8 France, 9Hong Kong, Kuwait, Singapore and Spain,5 and isolates resistant as a result of reduced carbapenem accumulation were found in the USA.10 Characterization of acinetobacter carbapenemases has been slow, owing to low yields, and comprehensive kinetics have only been reported for the enzyme from one French isolate, A. baumannii A1489 This was a zinc-independent enzyme with a high Vmax for oxacillin. The ARI-13 and ARI-24 carbapenemases, from isolates collected in Scotland and Argentina, respectively, have also been partially characterized. They were zinc-independent whereas the carbapenemase from a Cuban isolate was zinc-dependent,7 as are most efficient carbapenemases from other species.11In the present paper, we present the biochemical characterization of a carbapenemase found in A. baumannii isolates collected in Buenos Aires.
Materials and methods
Acinetobacter isolates
A. baumannii BA HCT 3, 15 and 19 were isolated in 1995 from separate patients at the Enrique Tornu Hospital, Buenos Aires. BA HCT 15 was from urine; the other isolates were from sputa. Species identification was by amplified ribosomal DNA restriction analysis12 and strain typing by random amplified polymorphic DNA (RAPD) analysis on a commercial system (Pharmacia, Milton Keynes, UK). As controls we used a collection of Acinetobacter spp. isolates assembled in 1984.13 This included organisms from London, Nottingham, Portsmouth (UK), Detroit (MI, USA) and Switzerland.
Antibiotics
Antimicrobials were from suppliers as follows: ampicillin and clavulanic acid (SmithKline Beecham, Brentford, UK); ceftazidime and cefuroxime (GlaxoWellcome, Stevenage, UK); cefotaxime (Roussel, Uxbridge, UK); piperacillin, tazobactam and tetracycline (Wyeth, Taplow, UK); cefoxitin and imipenem (Merck Sharp and Dohme, Hoddesdon, UK); amikacin, benzylpenicillin, cephaloridine, chloramphenicol, gentamicin, sulphamethoxazole and trimethoprim (Sigma, St Louis, MO, USA); co-trimoxazole, cephalothin and tobramycin (Lilly, Basingstoke, UK); meropenem (Zeneca, Macclesfield, UK), ciprofloxacin (Bayer, Newbury, UK); aztreonam (Bristol Myers Squibb, Syracuse, NY, USA); sulbactam (Pfizer, Sandwich, UK); and nitrocefin (BBL, Cockeysville, MD, USA).
Susceptibility tests
MICs were determined on IsoSensitest agar (Oxoid, Basingstoke, UK) with inocula of 104 cfu. Results were read after overnight incubation at 37°C as the lowest concentrations to inhibit growth completely.
Biological detection of ß-lactamase activity
Extracts were prepared by washing overnight growth from nutrient agar plates into 2 mL volumes of 0.1 M phosphate buffer, pH 7.0, and sonicating for 2 x 30 s at an amplitude of 12 µm. These extracts were mixed 1:1 with 40 mg/L antibiotic solutions in 0.1 M phosphate buffer and incubated for 1 h at 37°C. Subsequently, the mixtures were distributed into bore holes (0.7 cm diameter) cut in a bioassay plate containing 120 mL of Muelleragar (Oxoid) that had been spread with a 500-fold dilution of an overnight nutrient broth culture of Escherichia coli NCTC 10418. Inhibition zones were measured after over night incubation at 37°C.
Isoelectric focusing
Cell extracts were prepared as for the biological ß-lactamase assays, except that 0.01 M phosphate buffer, pH 7.0, was used; they were subjected to isoelectric focusing on gels containing equal mixtures of Resolyte pH 3.510 and Resolyte pH 48 (BDH, Poole, UK). Conditions were as described by Livermore & Williams,14 and the ß-lactamases were visualized with 0.5 mM nitrocefin.
ß-Lactamase induction assays
ß-Lactamase induction assays were performed as de scribed by Livermore & Williams14 with cefoxitin 50 mg/L as the inducer. Induced activity was assayed against 0.1 mM imipenem as described below.
ß-Lactamase purification
A 1 L overnight culture of isolate BA HCT 15 in Nutrient Broth No. 2 (Oxoid) was added to 10 L of fresh identical broth and incubated for 4 h at 37°C, with shaking. The cells were then harvested by centrifugation at 5000g for 30 min at 4°C, washed twice in 20 mM TrisHCl, pH 8.5, resuspended in 25 mL of the same buffer, and disrupted by three passes through a French Pressure cell (SLM Aminco, Urbana, IL, USA). Debris was removed by centrifugation at 100,000g for 45 min and 4°C and the supernatant was loaded on to a column (40 cm x 2.6 cm) of diethyl aminothyl Sephadex A-50 (Pharmacia) in 20 mM TrisHCl, pH 8.5. This column was washed with two to three volumes of this buffer, then eluted with the same buffer containing a linear gradient of 00.5 M NaCl. Fractions containing ß-lactamases were located with nitrocefin and tested against imipenem by spectrophotometry, as described below.
ß-Lactamase kinetics and inhibition assays
ß-Lactamase assays were performed by spectro photometry at 37°C in 0.1 M phosphate buffer, pH 7.0, at the wavelengths listed by Livermore & Williams. 14 Vmax and Km values were calculated from the Hanes plots of initial velocity data. Inhibition was examined under conditions (i) where the enzyme was incubated with inhibitor for 10 min at 37°C before the addition of cephaloridine as the substrate or (ii) where the inhibitor was added to a mixture of the enzyme and substrate.14
ß-Lactamase molecular weight
To determine its molecular weight, the ß-lactamase purified from strain BA HCT 15 was subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) and in-situ reactivation by the method of Tai et al.15 as adapted by Livermore et al.16 Activity was detected by overlaying the washed gel with 0.5 mM nitrocefin.16
Transfer and curing of resistance
Transfer of resistance to E. coli K-12 J-53 1 (pro Rifr) was attempted by plate mating.14 Curing was attempted by growing cultures overnight in nutrient broth containing ethidium bromide at 0.250.5 8 x MIC, then replica plating on to IsoSensitest agar (Oxoid) with and without imipenem 2 mg/L or 10 mg/L.
Results
Typing
Isolates BA HCT 3 and 15 appeared identical by RAPD analysis, whereas isolate BA HCT 19 was different (not shown).
Antibiotic susceptibility
MICs of imipenem and meropenem for isolates BA HCT 3, BA HCT 15 and BA HCT 19 were from 2 to 4 mg/L (Table I), whereas carbapenem MICs for control isolates ranged from 0.12 to 0.5 mg/L. The three Argentinian isolates were resistant to all other conventional antimicrobials except amikacin, but retained susceptibility to sulbactam 4 mg/L. Resistance to non-carbapenem drugs was widespread among the control isolates: only ciprofloxacin, tobramycin amikacin, ceftazidime and cefepime had MIC50s at or below BSAC breakpoints.17
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Extracts of isolates BA HCT 3 and BA HCT 15 yielded ß-lactamase bands with pIs of 9.5, 7.5, 6.9 and 6.0; extracts of strain BA HCT 19 gave bands with pIs of 9.5 and 7.3. Most of the control isolates had ß-lactamases with pIs > 9.0; some also had ß-lactamases with the pIs typical of TEM-1 and -2.
Biological detection of ß-lactamase activity
Crude extracts of the three Argentinian isolates were all active against imipenem, but not against meropenem. These extracts also were strongly active against cefotaxime, cefuroxime and aminopenicillins, but not against ceftazidime, cefepime or latamoxef. Extracts from the control isolates mostly had strong activity against the amino penicillins, cefoxitin and cefuroxime, but none had activity against either carbapenem.
Characteristics of the carbapenemase from A. baumannii BA HCT 15
Fractionation of the ß-lactamases from strain BA HCT 15 was achieved by anion exchange chromatography. Carbapenemase activity was associated only with the pI 6.9 enzyme, and its kinetics were studied in detail (Table II). Penicillins were the best substrates: the Vmax for oxacillin was more than double that for any other penicillin, but similar Vmax/Km ratios were found for amino penicillins, carbenicillin, oxacillin and benzylpenicillin. Cephaloridine and cephalothin were hydrolysed rapidly, but theirVmax/Km ratios were more than ten-fold lower than those for the penicillins. Oxyimino-aminothiazolyl cephalosporins appeared stable within the sensitivity limits of the assays. Hydrolysis of imipenem was slow withVmax and Vmax/Km both substantially lower than for cephaloridine. Meropenem hydrolysis was not detected. Clavulanic acid and tazobactam inhibited the ß-lactamase (Table III): inhibition by clavulanic acid was not increased by preincubation, indicating reversibility, but inhibition by tazobactam was increased by preincubation, indicating irreversibility. EDTA (5 mM) did not affect enzyme activity, indicating that the ß-lactamase was not a zinc type. Meropenem, 1 µM, achieved 50% inhibition of cephaloridine hydrolysis, indicating high affinity.
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Genetic studies
Transfer of imipenem resistance to E. coli J-53-1 from any of the three resistant isolates was not detected, and cured derivatives were not obtained, despite several attempts.
Discussion
Acinetobacter isolates increasingly show multi-resistance.2 The 1984 collection used as source of control strains was not assembled on the basis of antibiogram, yet most isolates were resistant or barely susceptible to all ß-lactams except carbapenems and sulbactam, and to all other antimicrobials except tobramycin, amikacin and ciprofloxacin. Because of such resistance, carbapenems have become the main option against Acinetobacter spp. infections, and the emergence of resistance is disturbing. In the present paper we describe a carbapenemase from one isolate (BA HCT 15) with low-level carbapenem resistance. Another resistant isolate (BA HCT 3) from the same hospital had DNA and ß-lactamase profiles identical to BA HCT 15, but a third (BA HCT 19) gave different profiles and its carbapenemase awaits study.
The carbapenemase from BA HCT 15 had a pI of 6.9, and was produced together with pI 9.5, 7.5 and 6.0 enzymes that lacked imipenemase activity. The pI 6.9 enzyme was predominantly a penicillinase, with oxacillin the best substrate in terms ofVmax, although benzyl penicillin was a better substrate in terms ofVmax/Km. Cephaloridine and cephalothin were ten-fold weaker substrates than the penicillins and the enzyme had minimal activity against oxyimino-aminothiazolyl cephalosporins. It was active against imipenem butdespite the resistance of the isolateapparently not against meropenem. Nevertheless, meropenem was tightly bound by the enzyme, implying that the slow turnover might be efficient under periplasmic conditions.18 Activity of the enzyme was weakly and reversibly inhibited by clavulanic acid, whereas tazobactam was a weak, irreversible inhibitor. This substrate and inhibition profile is similar to that of the carbapenemase fromA. baumanniiA-148, which was collected in France.9 Nevertheless, there were differences: first, the present enzyme had a pI of 6.9, compared with 6.3 for that from strain A148; secondly,Vmax rates of the present enzyme for oxacillin:penicillin G:cephaloridine were in the ratio 10:5:1.3, whereas hydrolysis rates for these three substrates were approximately equal with the enzyme from strain A148. The present enzyme may or may not correspond to ARI-2,4 which was also found in carbapenem-resistant Acinetobacter isolates from Argentina, or to ARI-1, from a Scottish isolate.3 The pIs reported for ARI-1 (6.65) and ARI-2 (6.5) are lower than for the present enzyme (6.9), but these differences are within experimental variation. Unfortunately, few kinetic data are available for ARI-1 and -2; in particular, their oxacillinase activity has not been reported and no kinetics have been published for imipenem hydrolysis. Unlike the present enzyme, ARI-1 and -2 were both coded for by plasmids that could be cured or transferred.3,4
The present enzyme had the characteristics of ß-lactamase group 2 in the functional classification,11 but its further placement is difficult. It could be assigned to group 2d, which comprises enzymes with highestVmax rates for oxacillins, or to group 2f, which comprises the few non-zinc ß-lactamases active against carbapenems. Neither placement is satisfactory: other 2d enzymes are not active against imipenem and other 2f enzymes are not active against oxacillin.11 ,19 Full classification must await sequencing, which will reveal whether this acinetobacter enzyme belongs to molecular class D, as do the other group 2d types, or to class A, which includes the present members of group 2f.
Acknowledgments
We are grateful to Jens Berlau from the Laboratory of Hospital Infection, CPHL for assistance with identification and RAPD techniques.
Notes
* Corresponding author. Tel +44-818-200-4400;
Fax: +44-181-200-7499; E-mail: dlivermore{at}phls.co.uk
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Received 11 May 1998; returned 2 July 1998; revised 15 July 1998; accepted 12 August 1998