And Latamoxef Resistance in Pmactamase- Derepressed Pseudomonas Aeruginosa
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Journal of Antimicrobial Chemotherapy (1987) 20, 7-13 'Covalent trapping' and latamoxef resistance in pMactamase- derepressed Pseudomonas aeruginosa D. M. Lfvennore Downloaded from https://academic.oup.com/jac/article/20/1/7/773707 by guest on 27 September 2021 Department of Medical Microbiology, London Hospital Medical College, Turner Street, London El 2AD, UK Three Pseudomonas aeruginosa strains which constitutively produced chromosomal (Id, or Sabath and Abraham) /Mactamase in large amounts were resistant to latamoxef (moxalactam) MICs, 128-256 mg/1). Their 0-lactamase-basal mutants, which produced 1200-18,000-fold less enzyme, were latamoxef-sensitive (MICs, 4-16 mg/1), suggesting that the enzyme caused the resistance of the parent organisms. Latamoxef was a feeble substrate of the enzyme (kM <0-5/min) but reacted to form a stable complex that lacked catalytic activity against benzylpenicillin. The complex was isolated by gel filtration and was shown to be stable to isoelcctric focusing, suggesting a covalent link between the enzyme and latamoxef. During incubation the complex underwent a slow breakdown, regenerating active enzyme. This breakdown obeyed first-order kinetics, and the half-life of the inactivated form was 19±lmin at 37°C. Binding of antibiotic molecules in this complex may contribute to the latamoxef-resistance observed in the /Mactamase-dereprcsscd strains. This 'covalent trapping' should be distinguished from the 'non-covalent trapping' proposed elsewhere as a general mechanism of /J-lactamase-mediated resistance to reversibly-bound weak-substrate /Mactams. Introduction Debate continues on the mechanism whereby production of large amounts of Class I /Mactamases can protect Gram-negative bacteria against newer /J-lactam antibiotics. The enzymes have low turnover numbers (Vmn or kal) for many third-generation cephalosporins, but exhibit high affinity (l°w ^m or ^i) f°r these antibiotics (Livermore, 1983, 1985; Sanders, 1983). It has been postulated that resistance may involve non-hydrolytic trapping of the antibiotic molecules by the /Mactamase but, increasingly, the importance of hydrolysis is agreed (Livermore, 1985; Vu & Nikaido, 1985; Sanders & Sanders, 1986). The high enzyme-antibiotic affinity serves to maintain the hydrolytic efficiency of the enzyme, even at the low antibiotic concentrations that may be obtainable in the cell periplasm (Livermore, 1983, 1985; Vu & Nikaido, 1985). A hydrolytic model of resistance seems less applicable to latamoxef (moxalactam), which is extremely stable to Class I /J-lactamases, than to cephalosporins such as cefotaxime and ceftriaxone, which are more labile (Livermore, 1985; Sanders & Sanders, 1986). Nonetheless, over-production of Class I /Mactamases can protect bacteria against latamoxef (Sanders, 1983; SandeTS and Sanders, 1986) and the basis for this behaviour was examined in Pseudomonas aeruginosa. 7 0305-7453/87/070007 + 07 $02.00/0 © 1987 The British Society for Antimicrobial Chemotherapy D. M. Livennore Materials and methods Strains P. aeruginosa strains PsSOSAI""1, M1405 and M2297 constitutivcly synthesized large amounts of the chromosomal Id (Sabath & Abraham) /Mactamase. Their 0-lactamase-basal mutants PsSOAI^'-def, M1405-def and M2297-def, which synthesized several thousand-fold lower levels of enzyme, were obtained by mutagenesis with W-methyl N' nitro N-nitrosoguanidine, using the method described Downloaded from https://academic.oup.com/jac/article/20/1/7/773707 by guest on 27 September 2021 by Curtis et al. (1978). The derivation and characterization of these strains and mutants have been described in detail elsewhere (Livennore & Yang, 1987). Antibiotics Latamoxef diammonium salt was obtained from Eli Lilly Inc., Indianapolis, IN, USA; benzylpenicillin sodium, cephaloridine and nitrocefin were obtained from Glaxo Group Research, Greenford, Middlesex, UK. Minimum inhibitory concentrations (MICs) MICs were determined in solid media as described previously (Livennore, Williams & Davy, 1985). fl-lactamase quantitation The amounts of /Mactamase produced by the various mutants were quantified by nitrocefin-hydrolysis assays, by the method of Jacobs, Livermore & Davy (1984). P-lactamase purification and assays /?-lactamase was extracted from P. aeruginosa PsSOSAI00"1 and purified to 90% homogeneity as described by Livermore et al. (1985). The enzyme quantities quoted below were based on this purity estimate, on determinations of protein concentration by the method of Lowry et al. (1951), and on a molecular weight estimate of 40,000 (Livermore et al., 1985). Enzyme activity was measured by UV spectrophotometric assay in 01M phosphate buffer pH 70 at 37°C. The wavelengths were 257 nm for latamoxef, 235 nm for benzylpenicillin and 295 nm for cephaloridine. Kinetic parameters (kM, VmMX and A^J were derived from Hanes (s/v vs s) plot of initial velocity (v) data at various substrate (s) concentrations. Isolation of the latamoxef-enzyme complex Enzyme (250 pmoles) and latamoxef (2-5 /xmoles) were incubated together in 250 /il of 01 M phosphate buffer pH 70 at 37°C for 15 min. The mixture then was chromatographed at 4°C on a column (30 x 0-9 cm) of Sephadex G-25 Medium Grade (Pharmacia Fine Chemicals, Uppsala, Sweden). Equilibration of this column was with 01 M phosphate buffer pH 70. Elution was with the same buffer at a flow rate of about 20 ml/h. Coraknt trapping of latamoxef 9 lsoelectric focusing of the latamoxef-enzyme complex Column-eluted latamoxef-enzyme complex and similarly-chromatographed latamoxef- untreated enzyme were subjected to isoelectric focusing in Ampholine PAG gels pH 3-5-9-5 (LKB Instruments, Bromma, Sweden) for 60 min at 15W constant power at ca. 10°C. After focusing, the proteins were precipitated by placing the gel for 1 h in an aqueous solution of 10% (w/v) trichloroacetic acid and 5% (w/v) sulphasalicylic acid. Excess precipitant was removed by soaking the gel in methanol: acetic acid: water (30: 10: 60) for 2 h, before staining the proteins in 0-2% (w/v) Co-omassie Blue in Downloaded from https://academic.oup.com/jac/article/20/1/7/773707 by guest on 27 September 2021 methanol: acetic acid: water (30:10:60) for 4h. The background gel then was decolourized with multiple washes of methanol: acetic acid : water (30 : 10 : 60). Results MICs The MICs of latamoxef for the /Mactamase constitutive (stably derepressed) strains and their basal-mutants are shown in Table I, together with quantitative data on /Mactamase production. All three enzyme-constitutive strains were substantially more resistant to latamoxef than their enzyme-basal mutants. fi-Lactamase assays 00111 /?-Lactamase from PS50SAI catalysed extremely slow hydrolysis of latamoxef: kat, the number of latamoxef molecules hydrolyzed per minute per molecule of enzyme, was estimated to be 0-5 or less, and derivation of J^ by direct assay was not possible. kM and Km values for cephaloridine were 17 000/min and 200 y.u, respectively; values for benzylpenicillin were 5300 min and 15 /iM, respectively. Latamoxef inhibited hydrolysis of cephaloridine or benzylpenicillin by the enzyme, and the kinetics of this process were investigated. In one type of experiment enzyme (ca 5 pmoles) was added to a mixture of 1 fiM latamoxef and 0-5 mM cephaloridine, and the decline in the cephaloridine concentration was monitored (Figure 1, B). Under these conditions the cephaloridine hydrolysis rate initially decelerated rapidly, halving before 20% of the cephaloridine had been consumed, then stabilized at an almost constant velocity. In a second type of experiment (Figure 1, Q, latamoxef (1 (IM) and Table I. MICs of /Mactams for P. aeruginosa strains and their mutants in relation to /Mactamase production Quantity of /J lactamase MIC latamoxef Strain U/mg cell protein" (mg/1) PsSOSAI00"' 21900 128 PsSOSAI000 '-def 17-2 4 M2297 10200 128 M2297-def 2-6 8 M1405 42100 256 M1405-def 0-5 16 *1 U hydrolysed 1 mnole nitrocefin/min at 37°C and pH 7-0 and at a nitrocefin concentration or 100 MM. 10 D. M. Ilvennore 0-5 Downloaded from https://academic.oup.com/jac/article/20/1/7/773707 by guest on 27 September 2021 5 10 Time (mln) Figure 1. Inhibition of cephaloridine hydrolysis by latamoxef. Hydrolysis of OS mil cephaloridine by 0-lactamase purified from PsioSAI""1 was measured under conditions: A, where no latamoxef was added to the mixture, B, where cephaloridine and I //M latamoxef were mixed and the reaction started by enzyme addition and C, where the enzyme and 1 /JM latamoxef were incubated together for 15 min at 37°C prior to initiation of hydrolysis by addition of cephaloridine. enzyme (ca. 5 pmoles) were incubated together in 1 ml volume for 15 min at 37°C, prior to the addition of sufficient cephaloridine (50 /il, 10 nui) to give a final concentration (0-5 HIM) identical to that in the non-pre-incubated system described above. Here the initial cephaloridine hydrolysis rate was slower than that seen in the non-pre-incubated system but rapid deceleration of the hydrolysis velocity was not observed during the first few minutes of the reaction. Characterization of latamoxef-enzyme complex The latamoxef-enzyme complex eluted with the void volume of the column, about 30 min after loading, and was separated completely from excess latamoxef, which eluted only after 4-5 h. Two protein species were detected when the complex was subjected to isoelectric focusing. These had pis of 8-2 and 7-6. The pi 8-2 species correspond to the antibiotic-untreated enzyme, whereas the pi 7-6 species was absent from the untreated enzyme preparation. The stability of the complex was examined by incubating the column-eluted material at 37°C and periodically adding 100 pi amounts to 1-ml volumes of 0-5 mM benzylpenicillin in 01 M phosphate buffer pH 70. Immediately after elution the complex lacked penicillin-hydrolysing activity. However, activity recovered progressively during incubation at 37°C and ultimately equalled that of column-passaged latamoxef-untreated enzyme.