3-Lactamase in Enterobacteria

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1984, p /84/ $02.00/0 Copyright 1984, American Society for Microbiology Vol. 25, No. 2 Detection of PSE-2 3-Lactamase in Enterobacteria DAVID M. LIVERMORE,* JEFFREY P. MASKELL, AND J. DAVID WILLIAMS Department of Medical Microbiology, London Hospital Medical College, London E.J., Great Britain Received 30 September 1983/Accepted 17 November 1983 PSE-2, a plasmid-mediated P-lactamase which was previously considered pseudomonas specific, was observed in clinical isolates of Escherichia coli, Klebsiella pneumoniae, Providencia stuartii, and Enterobacter cloacae. All four isolates transferred PSE-2 production, together with resistance to gentamicin, tobramycin, kanamycin, and sulfamethoxazole, into Escherichia coli J62-1 and Pseudomonas aeruginosa PU21. Transfer correlated with acquisition by the transconjugants of a 65-megadalton plasmid, and this element was considered to encode PSE-2 expression. The PSE-2 enzyme conferred high-level carbenicillin resistance on both J62-1 and PU21 transconjugants, although the PSE-2-producing Providencia stuartii isolate was susceptible to carbenicilin. The occurrence of plasmid-encoded P-lactamases in gramnegative bacteria was first recognized by Datta and Kontomichalou during the mid-1960s (4). Increasing dissemination of these enzymes during the ensuing two decades has been a major factor in increasing the resistance to antibiotics such as ampicillin and carbenicillin. At least 11 distinct plasmidmediated P-lactamases are currently recognized, distinguishable by their biophysical and catalytic properties (16). Their distribution reflects the properties of the elements on which their encoding genes may occur. Whereas the TEM enzymes occur widely in enterobacteria and Pseudomonas, Haemophilus, and Neisseria spp. and the OXA and SHV enzymes occur in the first two of these groups, the four PSE types have beeh considered to be restricted to Pseudomonas aeruginosa (16). Recent reports indicate the spread of the PSE-1 enzyme to Escherichia coli, Salmonella typhimurium, and Serratia liquefaciens (9, 14, 18). Transfer of the genes encoding the other PSE enzymes to non-pseudomonas hosts, has been achieved only by using recombinant plasmid techniques (6). The present- report describes detection of PSE-2 P-lactamase (15, 21) in four enterobacteria and an investigation of the properties of the element encoding enzyme production. Preliminary communication about PSE- 2 production by these organisms has been made (J. P. Maskell, D. M. Livermore, and J. D; Williams, Abstr. 13th Int. Cong. Chemother., Vienna, Austria, 1983). MATERIALS AND METHODS Bacterial strains. Escherichia coli R140, Klebsiella pneumoniae R156, Providencia stuartii R178, and Enterobacter cloacae R248 (the "R" strains) were isolated from separate patients at the London Hospital during , when a collection of 250 gentamicin-resistant organisms was being assembled. Strains R140, R156, and R178 were from patients in the same ward and were obtained within 3 months of each other. Precise source details on strain R248 can no longer be traced. Strains were identified by using the API 20E system and were maintained on nutrient agar slopes (Southern Group media, London) containing 2,ug of gentamicin per ml. Their production of PSE-2,-lactamase was revealed during a subsequent examination of the collection for,-lactamase synthesis and resistance to newer P-lactam antibiotics. The present study also included a spontaneous streptomycinsusceptible variant of strain R248 designated R248 Sms, * Corresponding author. which appeared during nutrient agar culture. The standard plasmid recipient organisms were an Escherichia coli J62-1 nalr mutant of strain J62 lac pro his trp (1) and Pseudomonas aeruginosa PU21 ilv leu str rif (7). Pseudomonas aeruginosa PU21, containing the PSE-2- coding R151 plasmid (3), was kindly provided by Glaxo Group Research, Greenford, England. Transconjugant preparation. Transconjugants of the R isolates and strain R248 Sms with Escherichia coli J62-1 and Pseudomonas aeruginosa PU21 were prepared by mixing overnight or late-logarithmic-phase broth cultures of the donor (0.5 ml) and recipient (4.5 ml) together with 5 ml of fresh nutrient broth (Southern Group media). After overnight incubation, 0.1 ml of the mixtures was spread across Oxoid Diagnostic Sensitivity Test (CM261) plates containing appropriate counterselective agents. These included carbenicillin (250 jig/ml) plus nalidixic acid (100,ug/ml) for R strain x J62-1 crosses and carbenicillin (250 plg/ml) plus rifampin (100 jig/ml) for R strain x PU21 crosses. In preliminary experiments, J62-1 transconjugants of the R strains were selected on Oxoid Diagnostic Sensitivity Test agar containing gentamicin (10 jig/ml) and nalidixic acid (100,ug/ml). During attempts to independently transfer a second,b-lactamase and streptomycin resistance plasmid from strain R248 (see below), mating mixtures of this organism with strain J62-1 were spread on Oxoid Diagnostic Sensitivity Test agar containing streptomycin (100,ug/ml) plus nalidixic acid (100 pug/ml). Counterselective plates were incubated at 37 C for up to 3 days, and the number of colonies was counted relative to the number of donor cells which could be recovered from the mating mixture. Ten presumptive transconjugants were selected from each mating and confirmed as P-lactamaseproducing, lactose nonfermenting auxotrophs (J62-1 transconjugants) or P-lactamase-producing, oxidase-positive auxotrophs (PU21 transconjugants). P-Lactamase production was tested as the ability of EDTA-lysozyme-disrupted cells to rapidly hydrolyze nitrocefin (13). Lactose fermentation was investigated by using MacConkey agar. Auxotrophy was tested as the inability of cells to grow on Kelly and Clarke mineral medium (10) solidified with 1% purified agar (Oxoid L28) and supplemented with 0.5% glucose. j3-lactamase extraction. 3-Lactamase extracts were prepared from the R strains and their J62-1 and PU21 transconjugants by either of two methods. Small amounts of enzyme for isoelectric focusing were obtained by washing overnight growth from nutrient agar slope cultures into 1-ml volumes 268

2 VOL. 25, 1984 of 0.1 M phosphate buffer (ph 7.0) and subjecting these suspensions to ultrasonication (Ultrasonic Disintegrator; MSE Instruments, Crawley, Sussex, Great Britain). Sonicates were cleared of larger cell debris by centrifugation at 12,000 x g for 15 min (Eppendorf 5414 centrifuge). Enzyme preparations for hydrolysis studies were prepared by sonication of late-logarithmic-phase cells harvested from nutrient broth (Oxoid no. 2 CM69) cultures and were cleared of membrane debris by centrifugation at 100,000 x g for 30 min at 4 C (MSE Superspeed 75 centrifuge). Isoelectric focusing of,3-lactamases. Isoelectric focusing of extracts was carried out in polyacrylamide gels according to the method of Matthew and colleagues (17). Enzymes were visualized by flooding the gel with 1 mm nitrocefin (19). Characterized f-lactamases, prepared from Escherichia coli and Pseudomonas aeruginosa strains and transconjugants provided by Glaxo Group Research, were included on these gels for comparison. 13-Lactamase hydrolysis studies. The hydrolytic activity of the,-lactamase extracts was investigated by spectrophotometric assay (20, 25). P-Lactam solutions were prepared at a concentration of 0.5 mm in 0.1 M phosphate buffer (ph 7.0), and absorbance changes after enzyme addition were monitored by using a Pye-Unicam SP 1700 spectrophotometer. The substrates and wavelengths used were as follows: benzylpenicillin, 235 nm; carbenicillin, 235 nm; ampicillin, 235 nm; oxacillin, 263 nm; cephaloridine, 295 nm; and methicillin, 305 nm. Enzyme inhibition by cloxacillin (0.1 mm) was tested by using benzylpenicillin (0.5 mm) as the substrate. Extracts of Pseudomonas aeruginosa PU21 transconjugants were used as sources of enzyme for these studies and were compared with extracts from strain PU21 (R151). Pseudomonas transconjugants were preferred to the wildtype R strains as sources of enzyme because the former did not produce substantial amounts of any secondary,b-lactamase. Furthermore, because all compared extracts were obtained from a standard host organism, the effects of any contaminant matter should have been standardized. Resistance phenotypes. MICs of amikacin, carbenicillin, chloramphenicol, gentamicin, kanamycin, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, tobramycin, and trimethoprim were determined for the R strains, strain J62-1, strain PU21, and 10 transconjugants from each mating. Doubling dilutions of antibiotics were incorporated into Oxoid Diagnostic Sensitivity Test plates, and inocula (ca. 104 CFU) were applied to the agar surface with a Denley multipoint device. Results were read after overnight incubation at 37 C, and the MIC was defined as the lowest antibiotic concentration preventing growth. Molecular weights of transferred plasmids. Molecular weights of plasmids transferred to the J62-1 and PU21 recipients were determined by the method of Birnboim and Doly (2). Electrophoresis was carried out in a 16-cm Protean electrophoresis tank (Bio-Rad Laboratories, Watford, Herts, United Kingdom) at a constant 160 V. Standards used as molecular weight markers were phh502 (70 megadaltons [Md]), phh502-1 (40 Md), cryptic plasmid (28 Md), phh500 (10 Md), phh501 (4 Md), and cryptic plasmid (2 Md). RESULTS 3-Lactamase production by R strains. Isoelectric focusing demonstrated that Escherichia coli R140, Klebsiella aerogenes R156, Providencia stuartii R178, and Enterobacter cloacae R248 synthesized P-lactamases which focused identically to that from strain PU21 (R151) at a pl of 6.1. Extracts PSE-2 1-LACTAMASE IN ENTEROBACTERIA 269 from strain R156 also contained a,-lactamase with a pi of 7.6 which aligned with the SHV-1 enzyme, and extracts from strain R248 contained an enzyme which cofocused with TEM-1 1-lactamase at a pi of 5.4 (Fig. 1). Additional 13- lactamase activities were detected in strains R178 and R248. These enzymes did not align with any of the plasmidmediated enzymes described by Matthew (16) and focused in the highly alkaline range (pl > 8.0) typical of the chromosomal 1-lactamases of gram-negative bacteria (24). The intensity of the bands given by these last enzymes was insufficient for resolution on Fig. 1. Strain R248 Sms produced the 6.1-pI (PSE-2-cofocusing) enzyme but did not synthesize the 5.4-pI (TEM-1-cofocusing) enzyme. Transfer of P-lactamase production. Resistance to carbenicillin was transferred by strains R140, R156, R248, and R248 Sms to strains J62-1 and PU21 at frequencies of ca and 10-7 per donor, respectively, when matings were performed with overnight cultures. These frequencies increased 10- to 100-fold when late-logarithmic-phase cultures were mated. Transfer frequencies from strain R178 were less than 10-9 per donor cell regardless of whether overnight or log-phase cells were mated, and only single transconjugants of this isolate with strains J62-1 and PU21 were obtained. Isoelectric focusing demonstrated acquisition of the 6. 1-pl 3-lactamase by all the J62-1 and PU21 transconjugants. Six of the 10 J62-1 transconjugants of strain R248 selected on carbenicillin-nalidixic acid agar also acquired synthesis of ph FIG. 1. Comparative isoelectric focusing of 1-lactamase from strains R140, R156, R178, and R248. Lane 1, TEM-1 enzyme; lane 2, extract from R248; lane 3, extract from R178; lane 4, PSE-2 enzyme from PU21 (R151); lane 5, extract from R140; lane 6, extract from R156; lane 7, SHV-1 enzyme; lane 8, TEM-2 enzyme.

3 270 LIVERMORE, MASKELL, AND WILLIAMS TABLE Lactamase activities of extracts of PU21 transconjugants of R strains and strain PU21 (R151) Relative hydrolysis rate (%)' Enzyme source Pen Cb Amp Oxa Meth Ceph PU21 (R140) PU21 (R156) PU21 (R178) PU21 (R248) PSE-2 control PU21 (R151) a Pen, Benzylpenicillin; Cb, carbenicillin; Amp, ampicillin; Oxa, oxacillin; Meth, methicillin; Ceph, cephaloridine. the TEM-1-cofocusing enzyme. All 10 J62-1 (R248) transconjugants selected on streptomycin-nalidixic acid agar acquired this,-lactamase in addition to PSE-2. Synthesis of the TEM-1-type enzyme was not acquired by PU21 transconjugants of strain R248. Transfer of,-lactamases other than PSE-2 from strains R140, R156, R178, and R248 Sms was not obtained with either recipient. Trace amounts of high-pi (>8.0) P-lactamases were detected in strains J62-1 R- and PU21 R- and in their transconjugants; these did not align with known plasmid-mediated enzymes and were considered to be of chromosomal origin. 3-Lactamase hydrolysis studies. The activity of the 6.1-pI enzyme extracted from the PU21 transconjugants of the R strains closely resembled that of the PSE-2 enzyme obtained from strain PU21 (R151) (Table 1). All these enzymes were more active, under the experimental conditions, against methicillin, ampicillin, and oxacillin than against benzylpenicillin. Carbenicillin was less rapidly hydrolyzed than benzylpenicillin, and cephaloridine was a very weak substrate. Cloxacillin did not inhibit hydrolysis of benzylpenicillin. Resistance phenotypes of R strains and their transconjugants. With the exception of strain R178, all the R isolates were resistant to carbenicillin (Table 2). They additionally possessed a broad-spectrum resistance to other antimicrobial agents. Strain R248 Sms differed from its parent organism in lacking streptomycin resistance and in having lower level resistance to gentamicin, tobramycin, and carbenicillin. J62-1 transconjugants of strains R140, R156, R178, and R248 Sms and PU21 transconjugants of all R strains acquired a constant pattern of resistance, encompassing carbenicillin, gentamicin, tobramycin, kanamycin, and sulfamethoxazole (Table 3). Aminoglycoside resistance was expressed at a higher level in the Pseudomonas aeruginosa transconjugants than in the Escherichia coli transconjugants. A more complex situation existed with the J62-1 (R248) transconjugants: of the 10 organisms selected on carbenicillin-nalidixic acid agar, the four which lacked the TEM-1 enzyme exhibited an antibiogram identical to that of the J62-1 transconjugants of the other donors. The other six TEM-1-containing, carbenicillin-nalidixic acid-selected J62-1 (R248) transconjugants, as ANTIMICROB. AGENTS CHEMOTHER. well as all 10 selected on streptomycin-nalidixic acid agar, also acquired streptomycin resistance and were more resistant to carbenicillin, gentamicin, tobramycin, and kanamycin. When gentamicin (10 p.g/ml)-nalidixic acid (100,g/ml) agar was used during the preliminary experiments for selection of J62-1 transconjugants of R strains, up to fourfold increases in amikacin resistance were observed in some of the transconjugants. This result was not reproduced when transconjugants were selected on carbenicillin-nalidixic acid agar. Gentamicin-nalidixic acid-selected transconjugants were also two- to fourfold more resistant to gentamicin and tobramycin than were carbenicillin-naxlidixic acid-selected organisms. Resistances to agents other than aminoglycosides were unaffected. We suggest that these anomalies derived from the high gentamicin concentration used in the counterselective plates and from a selection for transconjugants which also possessed elevated intrinsic resistance to gentamicin. Such resistance normally extends to most other aminoglycosides (22). Molecular weights of plasmids transferred. J62-1 and PU21 transconjugants of the R strains, including strain R248 Sms, acquired a 65-Md plasmid. Those J62-1 transconjugants of strain R248 which acquired TEM-1 enzyme production, streptomycin resistance, and higher level aminoglycoside and carbenicillin resistance (Table 3) were found to have further acquired a 30-Md plasmid. DISCUSSION Isoelectric focusing and hydrolysis studies found no significant differences between the 6.1-p1 enzyme expressed by the four isolates and the PSE-2 enzyme encoded by the R151 plasmid. We therefore deduce that these enzymes are identical and that PSE-2 can no longer be considered pseudomonas specific. The hydrolysis rates obtained do differ somewhat from those published by Matthew (15), who found PSE-2 to hydrolyze carbenicillin more rapidly than benzylpenicillin. It seems unlikely that these discrepancies were due to additional,b-lactamase activities in the unpurified PU21 extracts used, since electrofocusing detected only minimal contamination with a second high-pi enzyme which was probably of chromosomal origin. It is more likely that they depended on methodological differences; relative hydrolysis rates at single-substrate concentrations, although useful for enzyme comparison, represent an arbitrary measure of enzyme-substrate specificity (5). Considering the period ( ) when these organisms were isolated, it is apparent that the spread of the PSE-2 enzyme into enterobacteria is not a particularly recent event, and it seems likely that its initial classification as pseudomonas specific was erroneous. This classification was based solely on work with the R151 (3) plasmid, which at that time was the only known PSE-2-coding element. Other, widely TABLE 2. Antibiotic MICs for R strains MIC (1tg/ml)a Cb Gm Tb Km Sm Sp Ak Tc Tm Cm Su E. coli R140 > > >64 >64 16 >512 K. pneumoniae R156 > >64 >64 2 >512 P. stuartii R >128 >512 4 >64 64 >128 >512 E. cloacae R248 > > <1 <1 2 >512 E. cloacae R248 Sm' <1 <1 4 >512 a Cb, Carbenicillin; Gm, gentamicin; Tb, tobramycin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin; Ak, amikacin; Tc, tetracycline; Tm, trimethoprim; Cm, chloramphenicol; Su, sulfamethoxazole.

4 VOL. 25, 1984 PSE-2 1-LACTAMASE IN ENTEROBACTERIA 271 TABLE 3. Resistance transferred to strains J62-1 and PU21 ~~~~~Mol (~~~~~~giml)b wt Of Trans- Selection on MIC(3g/ml),-Lactamases plasmid(s) conjugant agar'3 acquired acquired Cb Gm Tb Km Sm Sp Ak Tc Tm Cm Su (Md) J62-1 C <4 J62-1 (R140) 10/10 nal-cb >512 PSE-2 65 J62-1 (R156) 10/10 nal-cb >512 PSE-2 65 J62-1 (R178) 1/1 nal-cb >512 PSE-2 65 J62-1 (R248) 4/10 nal-cb >512 PSE-2 65 J62-1 (R248) 6/10 nal-cb > >512 PSE-2, TEM-1 65, 30 J62-1 (R248) 10/10 nal-sm > >512 PSE-2, TEM-1 65, 30 J62-1 (R248 10/10 nal-cb >512 PSE-2 65 Sms) PU > PU21 (R140) 10/10 rif-cb >1024 >128 >128 >512 > >512 PSE-2 65 PU21 (R156) 10/10 rif-cb >1024 >128 >128 >512 > >512 PSE-2 65 PU21 (R178) 1/1 rif-cb >1024 >128 >128 >512 > >512 PSE-2 65 PU21 (R248) 10/10 rif-cb >1024 >128 >128 >512 > >512 PSE-2 65 PU21 (R248 10/10 rif-cb >1024 >128 >128 >512 > >512 PSE-2 65 Sins) a nal, Nalidixic acid; rif, rifampin; Cb, carbenicillin; Sm, streptomycin. b For definitions of abbreviations, see footnote a, Table 2. All MICs are subject to ±1 dilution. C Number refers to proportion of transconjugants tested with the stated antibiogram. disseminated P-lactamases such as TEM-1 have been observed on pseudomonas-specific plasmids (8). This wider distribution of PSE-2 is of some significance because its hydrolytic activity includes such newer 1-lactams as ceftriaxone, cefotaxime, and aztreonam, which are stable to most other plasmid-encoded P-lactamases (12, 23). PSE-2 enzyme production was apparently encoded by the 65-Md plasmid, which was acquired by all the transconjugants. This element additionally specified resistance to gentamicin, tobramycin, kanamycin, and sulfamethoxazole (Table 3) and differed from previously described PSE-2-coding plasmids (3, 21) both in its extended transmissibility and in its lack of spectinomycin and streptomycin resistance determinants. The absence of these last resistances is of some interest because the PSE-2 gene has recently been observed to occur on a transposon, Tn14O4, which encodes resistance to these aminoglycosides, as well as to carbenicillin, gentamicin, tobramycin, kanamycin, and sulfamethoxazole (21). TnJ404-determined spectinomycin and streptomycin resistance depends on a single adenyltransferase (G. A. Jacoby, personal communication). Our findings may indicate either that TnJ404 is not the sole PSE-2-encoding transposon or that the spectinomycin-streptomycin adenyltransferase gene of TnJ404 is defective in the present plasmids. The considerable carbenicillin resistance of J62-1 and PU21 transconjugants indicated efficient PSE-2 function in these hosts. This result supports the findings of Hedges and Matthew (6), who observed that R151, if transferred to Escherichia coli by a recombinant plasmid technique, afforded the recipient protection against P-lactams. Conclusions regarding PSE-2 function in the original isolates are more tenuous since the intrinsic 1-lactam resistance of these organisms is unknown. Furthermore, strains R156 and R248 possessed SHV-1 and TEM-1 P-lactamases, respectively, and these enzymes are known to confer carbenicillin resistance (12, 16). Interestingly, Providencia stuartii R178 was less resistant to carbenicillin than the other isolates, despite PSE-2 production (Table 2). This result may reflect the slightly lower level of enzyme expression by this organism, that its outer membrane is so permeable that the enzyme cannot function adequately to maintain the cell periplasm free of antibiotic, or that periplasmic conditions are unsuitable for efficient PSE-2 1-lactamase function. The host dependence of the aminoglycoside resistance encoded by the 65-Md plasmid was also of interest. This resistance, which may have depended on a single aminoglycoside-modifying enzyme, was expressed at a higher level in PU21 transconjugants than in J62-1 transconjugants. Host dependence of aminoglycoside resistance has been reported elsewhere (6) and may reflect differing levels of enzyme expression or the ability of a modifying enzyme to integrate into the host cytoplasmic membrane structure. The TEM-1-13-lactamase production, streptomycin resistance, and higher level gentamicin, tobramycin, and kanamycin resistances acquired by some J62-1 transconjugants of strain R248 were associated with a 30-Md plasmid (Table 3), and this element was deduced to code for these resistances. It was apparently nontransferable to strain PU21. Attempts to transfer this element to strain J62-1 independently by selection on streptomycin-nalidixic acid agar were unsuccessful; all transconjugants acquired the 65-Md plasmid. In conclusion, these studies indicate that PSE-2 production is not confined to Pseudomonas aeruginosa. A further study of 1,000 recently (1982) collected gram-negative bacteria from the London Hospital has detected enzymes cofocusing with PSE-2 in four more enterobacteria (one Escherichia coli, one Enterobacter cloacae, and two Klebsiella aerogenes) (S. Whitaker, P. Hajipieris, and J. D. Williams, Abstr. 13th Int. Congr. Chemother., Vienna, Austria, 1983, abstr. no. PS 2.5, p. 1-3), and work is in progress to confirm this identification. Despite these observations, we advocate retention of the term "PSE." There are already several systems of,-lactamase nomenclature, largely based on arbitrary criteria, and renaming is likely to increase the confusion. Ultimately it is hoped that a more fundamental classification, based on amino acid sequences and active-site structure (11), will be extended to encompass such enzymes as PSE-2. LITERATURE CITED 1. Bachmann, B. J Pedigrees of some mutant strains Escherichia coli K-12. Bacteriol. Rev. 36: of

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