Livermore, D. M., R. J. Williams, M. J. Lindridge, R. C. B.. Slack, and J. D. Williams. 1982. Pseudomonas aeruginosa iso- lates with modified beta-lactamase ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1986, 0066-4804/86/010099-05$02.00/0 Copyright © 1986, American Society for Microbiology
p.
Vol. 29, No. 1
99-103
Characterization of NPS-1, a Novel Plasmid-Mediated P-Lactamase, from Two Pseudomonas aeruginosa Isolates DAVID M. LIVERMOREt* AND CLIVE S. JONES The London Hospital Medical College, London El 2AD, United Kingdom Received 10 July 1985/Accepted 10 October 1985
A novel ,-lactamase, which had a pl of 6.5 and a molecular weight of 25,000, was observed in two Pseudomonas aeruginosa isolates. The enzyme, designated NPS-1, was encoded by a plasmid of molecular weight 41 x 106 which also encoded resistance to streptomycin and sulfonamide. This plasmid, designated pMLH50, was freely transmissible to other P. aeruginosa strains, but not to Escherichia coli K-12. The enzyme was purified partially and shown to have activity against both penicillins and cephalosporins. Vmax rates for oxacillin and carbenicillin were less than 50% of the Vmax for benzylpenicillin, and the Vmax for cephaloridine was only 3% of the Vmax for benzylpenicillin. Imipenem, aztreonam, and several antipseudomonal cephalosporins were stable to the enzyme. Hydrolysis of most substrates obeyed Michaelis-Menten kinetics, but cefsulodin induced a reversible reduction in the activity of the enzyme. Transconjugants of the ,I-lactamaseproducing isolates in P. aeruginosa PU21 acquired f(-lactam resistances which mirrored the hydrolytic activity of the enzyme.
P-Lactamases (EC 3.5.2.6) inactivate penicillins and cephalosporins by hydrolyzing the amide bond of the P-lactam ring and are a major source of resistance to these antibiotics. Some of the enzymes can be encoded by plasmids and are of especial concern since their increasing dissemination has introduced resistance in hitherto sensitive organisms. Matthew et al. (14) detailed 11 plasmid-mediated ,Blactamases from gram-negative bacteria, and this list has now been increased to include over 20 distinct enzymes (16, 20). Individual enzymes are distinguished by their biophysical and catalytic properties, and they are most readily typed by their isoelectric points (pls). Plasmid-mediated ,-lactamases are infrequent in Pseudomonas aeruginosa isolates from British hospitals (22, 23), but are an occasional source of high-level resistance to carbenicillin in this species. Recently we observed two P. aeruginosa strains which were highly resistant to carbenicillin (MIC, 2,048 pLg/ml) and which synthesized a P-lactamase with a pI of 6.5, distinct from previously described plasmidmediated P-lactamases (23). The present study characterized the biochemical properties of this enzyme and demonstrated that it was encoded by a self-transmissible plasmid. MATERIALS AND METHODS Bacterial strains. P. aeruginosa M302 and M335 were clinical isolates obtained at Barking Hospital, Barking, Essex, United Kingdom. Strain M302 was isolated from a leg ulcer on an outpatient who had previously undergone knee surgery at the hospital; strain M335 was a respiratory tract isolate from a traffic accident victim who had been admitted to the hospital's intensive-therapy unit. Despite these source differences, the organisms were of the same serotype (0:6) and had identical bacteriophage susceptibility profiles, and may therefore have represented duplicates. Both organisms were received by us during a recent national survey of antibiotic resistance in P. aeruginosa. During this survey
1,866 consecutive P. aeruginosa isolates (53 from Barking Hospital) were examined (22, 23). P. aeruginosa PU21 ilv leu rifstr was described by Jacoby (7), P. aeruginosa PA0674 met his ilv hsd rif was described by Dean et al. (5) and Escherichia coli K-12 J53-1 pro met nal and J53-2 pro met rif were described by Coetzee et al. (3). Reference P. aeruginosa strains, which produced known plasmid-mediated ,Blactamases (PSE-1, PSE-2, PSE-3, and LCR-1), were provided by Glaxo Group Research. Antibiotics and other reagents. Ampicillin, carbenicillin, and methicillin were obtained from Beecham Research Laboratories; cefsulodin was obtained from CIBA-GEIGY Pharmaceuticals; benzylpenicillin, ceftazidime, cephaloridine, nitrocefin, and streptomycin were obtained from Glaxo Group Research; cefotaxime was obtained from HoechstRoussel Inc.; tetracycline was obtained from Lederle Laboratories; moxalactam was obtained from Eli Lilly & Co.; cefoxitin and imipenem were obtained from Merck Sharpe and Dohme Research Laboratories; chloramphenicol was obtained from Parke Davis and Co.; cefoperazone was obtained from Pfizer Inc.; ceftriaxone was obtained from Roche Products Ltd.; aztreonam was obtained from E. R. Squibb & Sons; spectinomycin was obtained from Upjohn Ltd.; and sulfonamide was obtained from the Wellcome Foundation Ltd. Other reagents were obtained from British Drug Houses or Sigma Chemical Co. -Lactamase identification by analytical isoelectric focusing. ,-Lactamases were extracted from nutrient agar slope cultures as described previously (9). Isoelectric focusing was performed routinely overnight at low power (1 to 4 W) in 7% polyacrylamide gels containing 2% (vol/vol) Pharmalyte (pH 5 to 8) (Pharmacia Fine Chemicals) on a home-made apparatus by the method of Matthew et al. (15). Rapid focusing was performed occasionally, with a Pharmacia FBE 3000 apparatus at 20 W constant power for 90 min. The gels for this system were supplemented with 10% (wtlvol) sucrose but were otherwise identical to those used on the low-power system. ,B-Lactamases were visualized by flooding the gels with 1 mM nitrocefin. Transfer of resistances. Exponential-phase cultures (ca.
* Corresponding author. t Address until May 1986: Department of Microbiology, University of Hong Kong, Hong Kong.
99
100
LIVERMORE AND JONES
5 x 108 CFU/ml) of donor cells (0.5 ml) which had been grown in nutrient broth were mixed with 1.0-ml volumes of stationary-phase recipient cultures (ca. 2 x 109 CFU/ml), which had been grown in the same medium. The cells in these mixtures were harvested immediately in an Eppendorf tube, suspended in 50 ,lI of nutrient broth, and transferred to a membrane filter (pore size, 0.45 p.m; Millipore Corp.), which was placed on a nutrient agar plate and incubated at 37°C for 2 or 18 h. The filter then was transferred to 3 ml of fresh nutrient broth and agitated to wash off the cells. The washings were diluted serially and spread on to Diagnostic Sensitivity Test Agar (CM261; Oxoid Ltd.) containing carbenicillin (250 ,ug/ml) or streptomycin (200 jig/ml) plus rifampin (200 ,ug/ml). Determination of plasmid molecular weight. Plasmid DNA from strains M302 and M335 and their transconjugants was extracted and visualized as described previously (10), except that a horizontal slab gel apparatus was used. MICs of antibiotics. MICs were determined by the agar dilution method, with doubling dilutions of antibiotics in Diagnostic Sensitivity Test Agar. Inocula contained ca. 104 CFU. I8-Lactamase extraction and purification. Strain M302 was used as a source of enzyme for these studies. Lateexponential-phase cells were harvested from 2 liters of nutrient broth (CM69; Oxoid) culture, suspended in 25 ml of 0.01 M phosphate buffer (pH 7.0), and frozen and thawed twice to liberate the periplasmic contents. Debris was removed by centrifugation at 100,000 x g (30 min at 4°C), and the ,B-lactamase-containing supernatant was subjected to preparative isoelectric focusing for 6 h at 30 W in Ultrodex (LKB Instruments Ltd.) containing 2% (vol/vol) Pharmalyte (pH 5 to 8). A Pharmacia FBE 3000 apparatus was used for this electrophoresis. The focused enzyme was located with nitrocefin and eluted into 0.05 M phosphate buffer (pH 7.0). ,B-Lactamase assays. ,B-Lactamase activity was measured against various substrates in 0.1 M phosphate buffer (pH 7.0) at 37°C by UV spectrophotometric assay (17, 21). Reaction mixtures contained a 1.1-ml volume in a cuvette with a light path of 1 cm. The wavelengths used were as follows: aztreonam, 208 nm; ampicillin, azlocillin, benzylpenicillin, and carbenicillin, 235 nm; cephaloridine, 255 and 295 nm; cefotaxime, ceftazidime, ceftriaxone, and moxalactam; 257 nm; cefsulodin and oxacillin, 263 nm; cefoperazone, 265 nm; imipenem, 297 nm; and methicillin, 305 nm. Kinetic constants were derived by linear regression analysis of Hanes plots of initial-velocity data at different substrate concentrations (4). The pH optimum was determined with 0.5 mM benzylpenicillin as the substrate in various 0.1 M phosphate buffers. I-Lactamase inducibility. Carbenicillin, cephaloridine, and cefoxitin at concentrations of 500 Vig/ml were tested as inducers of ,B-lactamase by the method described in reference 12. The 1-lactamase activity of sonicated induced and uninduced cultures was measured against 1 mM carbenicillin and 1 mM cephaloridine by spectrophotometric assay and was standardized against protein concentration, which was determined by the Lowry method (13). fI-Lactamase molecular weight. The molecular weight of the 3-lactamase was estimated by gel filtration (1) of the partially purified enzyme on a column of Sephadex G1SOSF (Pharmacia Fine Chemicals) which had been equilibrated in 0.05 M phosphate buffer (pH 7.0) and which was eluted with the same buffer. Standards used, and their molecular weights, were as follows: bovine serum albumin (68,000),
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Isoelectric focusing of NPS-1 13-lactamase. Lanes: 1, PSE-1; 2, LCR-1; 3, PSE-2; 4, extract from strain M302; 5, extract from a M302 x PU21 transconjugant; 6, extract from M335; 7, extract from a M335 x PU21 transconjugant; 8, PSE-3 P-lactamase.
ovalbumin (45,000), pancreatic DNase (30,000), myoglobin (17,800), and RNase (13,000). RESULTS Isoelectric focusing. Isoelectric focusing at low power (1 to 4 W overnight) confirmed that P. aeruginosa M302 and M335
synthesized a 1-lactamase (designated NPS-1) which focused between PSE-2 and PSE-3 enzymes, at a pl of about 6.5 (Fig. 1). This enzyme focused at a slightly lower pl (ca. 6.0) than did PSE-2 under high-power (20 W for 90 min) focusing conditions, although the reason for this anomaly is unclear. Trace quantities of a second 1-lactamase, which had a pl of >8.0, were present in extracts from strains M302 and M335, and this was considered to be the chromosomal Sabath and Abraham cephalosporinase, which is produced by virtually all P. aeruginosa strains (6). Transfer of resistances. Strains M302 and M335 both transferred carbenicillin, streptomycin, and sulfonamide resistance to P. aeruginosa PA0674 at frequencies of 10-' to 10-5 per donor cell. Transfer of carbenicillin resistance to P. aeriuginosa PU21 occurred at a similar frequency; the acquisition of streptomycin resistance could not be monitored in this organism, which was intrinsically resistant to this antibiotic. Both the PA0674 and the PU21 transconjugants were confirmed to have acquired production of NPS-1 Ilactamase. This is illustrated, for the PU21 transconjugants, in Fig. 1. The donor strains were also highly resistant to
spectinomycin (MIC, >1,000 ,ug/ml), chloramphenicol (MIC, >200 ,ug/ml), tetracycline (MIC, >200 ,ug/ml), and mercuric chloride (MIC, >50 ,ug/ml), but these resistances
were not transferred. The NPS-1 coding element did not alter the MICs of amikacin, gentamicin, tobramycin, and spectinomycin for the transconjugants. Transfer of carbenicillin resistance to E. coli K-12 recipients was not obtained. A 41-megadalton plasmid was detected in both donors and appeared in their transconjugants. This element, which we designate pMLH50, was inferred to encode the carbenicillin, streptomycin, and sulfonamide resistances.
NPS-1 3-LACTAMASE
VOL. 29, 1986 TABLE 1. Activity of NPS-1 ,B-lactamase from P. aeruginosa M302 and its contribution to resistance Antibiotic
Benzylpenicillin Ampicillin Oxacillin Cephaloridine Methicillin Carbenicillin Azlocillin Cefsulodin Cefoperazone Imipenem Cefotaxime Ceftriaxone Ceftazidime Moxalactam Aztreonam
Activity KVmax, Vmax Kill Vmax(pM) K,,, h
100 223 40d 3
