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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1990, p. 755-758 Vol. 34, No.

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0066-4804/90/050755-04$02.00/0
Copyright © 1990, American Society for Microbiology

Biochemical Characterization of a P-Lactamase That Hydrolyzes


Penems and Carbapenems from Two Serratia marcescens Isolates
YOUJUN YANG,t* PEIJUN WU, AND D. M. LIVERMORE
Department of Medical Microbiology, The London Hospital Medical College, Turner Street,
London El 2AD, United Kingdom
Received 30 June 1989/Accepted 18 January 1990

Reexamination of Serratia marcescens isolates obtained in 1982 revealed two organisms that were resistant
to the penem FCE 22101 (MIC, 512 ,ug/ml) and imipenem (MIC, 16 ,ug/ml) and that had slightly reduced
susceptibilities to meropenem (MIC, 0.12 ,ug/mI). MICs of these agents for typical S. marcescens isolates were
1 to 8, 0.25 to 0.5, and 0.03 ,ug/ml, respectively. The two isolates were fully susceptible to broad-spectrum
cephalosporins, and only one was highly resistant to ampicillin and carbenicillin (MICs, >1,024 ,ug/ml). Both
isolates had 3-lactamases that focused at pls 8.2 and 9.7. The penicillin-resistant isolate additionally produced
the TEM-1 enzyme. The enzymes with pls of 8.2 and 9.7 were separated by cation-exchange chromatography.
The pl 8.2 ,-lactamase was a class I enzyme of the type found in most S. marcescens isolates. It was almost
inactive against carbapenems and penems, as was the class I enzyme from another S. marcescens strain. The
pI 9.7 enzyme hydrolyzed penems and carbapenems rapidly: kCat (turnover number) values for FCE 22101,
imipenem, and meropenem were 3.4, 26, and 1% of the kcat value for cephaloridine, respectively; kciitIKm
values were 140, 915, and 150% of the kcaI/Km value for cephaloridine, respectively. Otherwise, the p1 9.7
enzyme had predominantly penicillinase activity. It was inhibited more readily by clavulanate than by
tazobactam and was inactivated by the chelating agents EDTA and ethylene glycol-bis(Ol-aminoethyl ether)-N,
N,N',N'-tetraacetic acid. Expression of the pI 9.7 enzyme was not associated with any plasmid, and production
was not transferred to Escherichia coli K-12 recipients, even after the mobilizing plasmid pUZ8 was inserted
into the S. marcescens donor strains.

Serratia marcescens isolates usually produce a chromo- mutant of strain S7 that was derepressed (17) for the class I
somal class I ,-lactamase with predominantly cephalospori- ,B-lactamase. Escherichia coli K-12 mutants J53-1 (pro met
nase activity (11). Expression of this enzyme generally is nal) and J53-2 (pro met rit) were described by Coetzee et al.
inducible but may become stably derepressed via mutation. (4). Plasmid pUZ8, which encodes resistance to kanamycin,
When the enzyme is inducible it confers resistance to those tetracycline, and mercurial compounds, was described by
drugs that are both labile and strong inducers of its synthe- Hedges and Matthew (5).
sis, for example, cephaloridine and ampicillin. Stable dere- Antibiotics. The following drugs were obtained from the
pression causes additional resistance to drugs that are labile indicated suppliers: mezlocillin, Bayer UK Ltd. (Newbury,
but that are weak inducers, including many broad-spectrum England); ampicillin, carbenicillin, and clavulanate,
cephalosporins (17, 17a). The enzyme lacks significant ac- Beecham Pharmaceuticals (Brentford, England); tazobac-
tivity against penems and carbapenems such as FCE 22101, tam, Cyanamid International (Gosport, England); FCE
imipenem, and meropenem (17, 17a) and does not provide 22101, Farmitalia Carlo Erba (St. Albans, England); benzyl-
the organism with resistance to these agents, irrespective of penicillin and cephaloridine, Glaxo Group Research (Green-
its mode of expression. Plasmid-mediated P-lactamases, ford, England); meropenem, ICI Pharmaceuticals (Maccles-
particularly the TEM-1 enzyme, also occur frequently in S. field, England); cefoxitin and imipenem, Merck Sharp &
marcescens strains (12), causing resistance to penicillins and Dohme Ltd. (Hoddesdon, England); ceftriaxone, Roche
older cephalosporins, but not to penems, carbapenems, or Products Ltd. (Welwyn Garden City, England); cefotaxime,
most newer cephalosporins. Roussel Laboratories Ltd. (Wembley, England); and nali-
In the present report we describe the biochemical proper- dixic acid and rifampin, Sigma Chemical Co. (St. Louis,
ties of an unusual enzyme that hydrolyzes penems and Mo.).
carbapenems which was observed in two S. marcescens Isoelectric focusing. Crude P-lactamase extracts were pre-
isolates that were resistant to imipenem and FCE 22101. pared as described by Livermore and Jones (8). Crude
MATERIALS AND METHODS P-lactamase extracts or purified preparations (see below)
were subjected to isoelectric focusing on pH 3.5 to 9.5
Bacterial strains. All the S. marcescens strains used in this polyacrylamide gels (Pharmacia, Uppsala, Sweden) for 2 h at
study, including S6, S7, and S8, were isolated from separate 20 W of constant power on a flatbed apparatus (FBE-3000;
patients at The London Hospital during 1982. These organ- Pharmacia). The P-lactamases were visualized by overlaying
isms were identified by the API 20E system (La Balme Les the gel with nitrocefin (0.01% [wt/vol]) in 0.1 M phosphate
Grottes, Montalier, France). S. marcescens S7-con was a buffer (pH 7.0).
MICs. MICs were determined on Diagnostic Sensitivity
*
Corresponding author. Test (DST) agar (Oxoid Ltd., Basingstoke, England) with
t Present address: Infectious Disease Unit, Department of Med- inocula of 104 CFU.
icine, Massachusetts General Hospital East, 149 13th Street, P-Lactamase extraction and purification. The chromo-
Charlestown, MA 02129. somal P-lactamase of strain S7-con was purified as described
755
756 YANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. MICs of P-lactams for S. marcescens isolates


MIC (,ug/ml) for the following isolatesa:
Antibiotic P-Lactamase-inducible isolates
S6 S8 S7-con lacking plasmid-mediated
,B-lactamases (n = 4)
Ampicillin 32 2,048 256 8-16b
Carbenicillin 32 1,024 32 2-4
Mezlocillin 4 64 64 1-2
Temocillin 16 16 8 8-16
Cephaloridine 512 1,024 256 128-256
Cefotaxime 0.25 0.25 4 0.12-0.5
Ceftriaxone 0.12 0.12 2 0.06-0.12
Cefoxitin 16 16 8 16-32
Imipenem 16 16 0.25 0.25-0.5
Meropenem 0.12 0.12 0.03 0.03
FCE 22101 512 512 2 1-4
3-lactamases
a The pIs of the P-lactamases from strain S6 were 8.2 and 9.7; those of the from strain S8 were 8.2, 9.7, and 5.4 (the pl 5.4 enzyme was TEM-1);
that of the ,-lactamase from mutant S7-con was 9.0; and those of P-lactamase-inducible isolates lacking plasmid-mediated 1-lactamases varied between 9.0 and
9.4.
b MIC range.

previously (17). To prepare the enzymes from strain S6, enzyme (25 pmol) and inhibitor was incubated at 37°C in 0.1
overnight cultures were grown in nutrient broth (no. 2; M phosphate buffer (pH 7.0). Samples (25 ,ul) were taken at
Oxoid) and were then diluted 10-fold into 8 liters of the same 0, 2, 5, 10, 20, 30, and 60 min after mixing and were added to
broth that was previously warmed to 37°C. The diluted 1-ml amounts of warm (37°C) 1 mM cephaloridine in the
cultures were incubated at 37°C with shaking for 4 h. The same buffer. The initial hydrolysis rate of the cephaloridine
cells were then harvested by centrifugation at 5,000 x g for was then measured by UV spectrophotometry.
15 min at 37°C, washed once with 0.1 M phosphate buffer ,-Lactamase induction assays. 1-Lactamase inducibility
(pH 7.0), and suspended in the same buffer at 200 times their was assayed routinely as described previously (18). Imi-
original density. P-Lactamases were released by subjecting penem (1 or 10 ,ug/ml), ampicillin (10 or 100 p.g/ml), and
these suspensions to four alternate cycles of freezing and cefoxitin (1 or 10 p.g/ml) were tested as inducers for strain
thawing. The crude enzyme extract thus obtained was puri- S6. The activity of enzyme extracts was measured against 10
fied by cation-exchange chromatography on a column (46 by mM cephaloridine or 1 mM imipenem.
2.8 cm) of carboxymethyl-Sephadex C-50 (Pharmacia). The Genetic studies. S. marcescens S6 and S8 were examined
column was equilibrated in 10 mM phosphate buffer (pH 8.2) for plasmids by the methods of Kado and Liu (7) and Bennett
and eluted with a gradient of sodium chloride (0 to 0.5 M) in et al. (1). Direct transfer resistance to E. coli K-12 deriva-
the same buffer. tives was attempted by the plate mating method described by
The purities and molecular weights of the enzymes were Livermore et al. (9). Transconjugant selection was done on
examined by sodium dodecyl sulfate-polyacrylamide gel DST agar containing FCE 22101 or imipenem (10 or 25
electrophoresis on the gel system of Lugtenberg et al. (10). ,ug/ml) plus nalidixic acid (100 p.g/ml) or rifampin (100
The molecular weight standards were phosphorylase b ,ug/ml), as appropriate. Selection of transconjugants of S8
(92,500), bovine serum albumin (68,000), ovalbumin was also attempted on DST agar containing carbenicillin (100
(45,000), carbonic anhydrase (31,000), soybean trypsin in- p.g/ml) plus nalidixic acid or rifampin (100 ,ug/ml).
hibitor (21,000), and lysozyme (14,400). Mobilization of the resistance determinants from S. mar-
I-Lactamase assays. Assays of P-lactamase activity were cescens S6 was attempted with plasmid pUZ8. This plasmid
performed by UV spectrophotometry with freshly prepared was transferred into strain S6 from an E. coli K-12 J53-2
antibiotic solutions in 0.1 M phosphate buffer (pH 7.0). The donor by plate mating as described above. Transconjugants
assays were run at 37°C, and the following wavelengths were were selected on DST agar containing kanamycin (100
used: 235 nm for benzylpenicillin, ampicillin, carbenicillin, p.g/ml) plus FCE 22101 (25 ,ug/ml). The transconjugants were
and mezlocillin; 255 nm for cefotaxime and FCE 22101; 257 then used as donors in crosses with the E. coli K-12
nm for ceftriaxone; 260 nm for cefoxitin; 295 nm for cepha- recipients.
loridine; and 297 nm for imipenem and meropenem. Values
of Km and VmS, were derived by linear regression analysis of RESULTS
Hanes or Lineweaver-Burk plots of initial velocity data that Antibiotic susceptibility. S. marcescens S6 and S8 were
were obtained at about 10 different substrate concentrations unusually resistant to FCE 22101 and imipenem compared
that ranged from 5 to 100 ,uM for the newer cephalosporins, with the other isolates collected in the same period (Table 1).
carbapenems, and penem and from 25 to 1,000 p.M for the Meropenem susceptibility was slightly reduced. S. marces-
penicillins and cephaloridine. The turnover number (kcat) cens S8 was additionally resistant to all penicillins except
was derived by dividing Vmax by the number of moles of the temocillin. Neither S6 nor S8 was resistant to broad-spec-
enzyme present. Since imipenem is chemically unstable, its trum cephalosporins compared with the resistances of con-
natural breakdown without enzyme was measured at each trol isolates that had only inducible expression of class I
substrate concentration. This rate was subtracted from the enzymes. Resistance to broad-spectrum cephalosporins and
hydrolysis rate observed in the presence of enzyme. all penicillins except temocillin was noted in the derepressed
Inhibition assays. Inactivation of the 3-lactamases by mutant S7-con, although this organism remained fully sus-
tazobactam and clavulanate was studied. A mixture of ceptible to the penem and carbapenem antibiotics.
VOL. 34, 1990 PENEM- AND CARBAPENEM-HYDROLYZING ENZYME 757

TABLE 2. Kinetic parameters of the pl 9.7 enzyme from S. marcescens S6 and the chromosomal enzyme from S7-con
pl 9.7 enzyme S7-con
Antibiotic
kcat (min-') Km (puM) km,, (min1) Km (pLM)
Benzylpenicillin 214 66 300 28
Ampicillin 2,700 600 7.7 92.6
Carbenicillin 58 25 2.3
Mezlocillin 26 1.1 7.8 24.5
Cephaloridine 2,500 1,100 19,000 2,080
Cefotaxime 38 20 19.2 5.2
Ceftriaxone 53 18 40.7 5.7
Cefoxitin 44 35 19.2
Imipenem 666 32 1.5 18
Meropenem 24 7 4.4 (f), 0.35 (s)a
FCE 22101 84 27 1.5 4.2
a Values for the fast (f) slow (s) phases of biphasic hydrolysis are given (17a).

Isoelectric focusing. Isoelectric focusing revealed that The pl 9.7 enzyme was inhibited by 1 mM EDTA or
strains S6 and S8 both had major P-lactamase activities ethylene glycol-bis(,3-aminoethyl ether)-N,N,N',N'-tetra-
focusing at pIs 8.2 and 9.7. Strain S8 additionally had an acetic acid (EGTA), 100 mM zinc sulfate, and 1 mM EDTA
enzyme that cofocused with the TEM-1 ,-lactamase at pH or EGTA mixed with 10 or 100 mM zinc sulfate or magne-
5.4. The inducible class I P-lactamases of the reference S. sium sulfate. Alone, 100 mM magnesium sulfate did not
marcescens strains gave single major bands at a scatter of pl inhibit the 1-lactamase.
values from 9.0 to 9.4. The enyzmes of S7 and its mutant P-Lactamase induction. Growth of strain S6 in the pres-
S7-con focused at pl 9.0 (Table 1). ence of ,B-lactams led to increased cephaloridine-hydrolyzing
Purification of ,-lactamases from S. marcescens. Ion-ex- activity in cell extracts but not to increased activity against
change chromatography separated the pI 8.2 and 9.7 ,B- imipenem. This suggests the induction of the pI 8.2 class I
lactamases that were produced by strain S6. The latter enzyme but not that of the pI 9.7 enzyme.
enzyme was more than 90% homogeneous as eluted from the Genetic studies. No plasmid was detectable in strain S6 by
column and was not purified further. Sodium dodecyl sul- either test method. A 28-kilobase plasmid was detected in
fate-polyacrylamide gel electrophories indicated that its mo- strain S8 by both methods. Attempts to transfer penem and
lecular weight was 25,000; that of the S7-con enzyme was carbapenem resistance from S6 and S8 were uniformly
43,000. unsuccessful, even when the mobilizing plasmid pUZ8 was
Hydrolysis of P-lactam antibiotics by S. marcescens I- inserted into the donor strains. Transconjugants of E. coli
lactamases. The pl 8.2 and 9.7 enzymes purified from strain K-12 recipients with the 28-kilobase plasmid from S8 were
S6 differed markedly in their hydrolytic properties. The obtained following selection with carbenicillin plus nalidixic
former enzyme (which was not purified to homogeneity) acid or rifampin. These transconjugants acquired the TEM-1
appeared similar to the chromosomal class I enzyme of enzyme but not the pl 9.7 enzyme.
S7-con, having a relative Vmax for benzylpenicillin that was
1.8% of the Vmax for cephaloridine. Vmax rates for ampicillin, DISCUSSION
mezlocillin, FCE 22101, and imipenem were <0.1% of that
for cephaloridine. The pl 9.7 enzyme, on the other hand, S. marcescens isolates typically have a chromosomal
differed radically from the S7-con enzyme (Table 2), being ,-lactamase of the Richmond and Sykes class I type. The
strongly active against FCE 22101 and imipenem and mod- ,-lactamase extracted from strain S7-con was representative
erately so against meropenem. Carbenicillin, mezlocillin, of this type. The pl 8.2 enzymes of S. marcescens S6 and S8
and especially ampicillin also were better substrates for the also appeared to be of the normal class I enzyme type,
pl 9.7 enzyme than for that of S7-con, whereas cephaloridine although their pl values were lower than that which is typical
showed the converse behavior. Benzylpenicillin was simi- (9.0 to 9.4; Table 1) of this species. The pI 9.7 enzymes of
larly labile to both enzymes. these isolates, on the other hand, behaved differently from a
Inhibition of pI 9.7 and S7-con enzymes. Inactivation of the class I enzyme, being of lower molecular weight and having
pl 9.7 enzyme by tazobactam or clavulanate was progres- significant activity against imipenem and FCE 22101. The
sive. The plot of the logarithm of residual enzymatic activity two strains with the enzyme expressed resistance to these
was linear with time, indicating that the inactivation was a last two compounds, but they were only marginally less
first-order process that could be described by the equation: susceptible than nonproducers to another carbapenem,
A = Aoe7kf, where Ao was the initial enzyme activity meropenem, which was a weaker substrate (lower kcat or
(defined as 100%) and A was the activity at time t; k was kcatlKm; Table 2). The pl 9.7 enzyme had some activity
variable with the inhibitor concentration. against several broad-spectrum cephalosporins, but the
The half-lives (t112) of enzymatic activity in the presence of MICs of these agents were not elevated for the enzyme
0.5 or 5 ,uM clavulanate (2 and 0.7 min, respectively) were producers.
shorter than that with the same concentrations of tazobac- Penem- and carbapenem-hydrolyzing activity is an un-
tam (9.5 and 3 min, respectively), indicating that clavulanate usual property among f-lactamases (3) that is shared by the
was the more potent inhibitor. The class I enzyme from chromosomal P-lactamases of a few species and a scattering
strain S7-con, like most class I ,-lactamases, is more sus- of unusual enzymes reported from occasional isolates. The
ceptible to inhibition by tazobactam than it is to inhibition by chromosomal enzymes that are active against carbapenems
clavulanate (M. Akova and D. M. Livermore, unpublished include those of Bacillus cereus, Flavobacterium odoratum,
data). and Pseudomonas maltophilia (2, 3, 13, 14). These enzymes
758 YANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.

appear to be structurally remote from one another (3) but The production and molecular properties of the zinc P-lacta-
share the property of having a zinc ion at their active sites. mase of Pseudomonas maltophilia IID 1275. Biochem. J. 229:
This ion is essential for catalytic activity. Zinc dependence is 791-797.
uncommon among P-lactamases, most of which function by 3. Bush, K. 1989. Classification of 3-lactamases: groups 2c, 2d, 2e,
another mechanism that entails the formation of an unstable 3, and 4. Antimicrob. Agents Chemother. 33:271-276.
4. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R-factors from
acyl ester between the free hydroxyl group of a serine at the Proteus rettgeri. J. Gen. Microbiol. 72:349-355.
active site and the carboxyl group of the opened ,-lactam 5. Hedges, R. W., and M. Matthew. 1976. Acquisition by Esche-
ring (3). richia coli of plasmid borne ,3-lactamases normally confined to
Imipenem hydrolysis has also been reported for occa- Pseudomonas spp. Plasmid 2:269-278.
sional isolates of S. marcescens and Enterobacter cloacae 6. Jarlier, V., M.-H. Nicholas, G. Fournier, and A. Phllippon. 1988.
(A. A. Medeiros and R. S. Hare, Program Abstr. 26th Extended broad-spectrum 1-lactamases conferring transferable
Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. resistance to newer 3-lactam agents in Enterobacteriaceae:
116, 1986), Aeromonas hydrophila (15; S. Bakken, C. C. hospital prevalence and susceptibility patterns. Rev. Infect.
Sanders, R. B. Clark, and J. P. Iaconis, 28th ICAAC, abstr. Dis. 10:867-878.
7. Kado, C. I., and S. T. Liu. 1981. Rapid procedure for detection
no. 495, 1988), and Bacteroides fragilis (19; G. J. Cucharal, and isolation of large and small plasmids. J. Bacteriol. 145:
S. Hurbut, and F. K. Tally, 26th ICAAC, abstr. no. 511, 1365-1373.
1986; C. Fijalkowski, F. Lamothe, F. Malounin, A.-M. 8. Livermore, D. M., and C. S. Jones. 1986. NPS-1: a novel
Bourgault, and L. Delorme, 26th ICAAC, abstr. no. 512, plasmid-mediated 1-lactamase from two isolates of Pseudomo-
1986). The genetic basis of production of these imipene- nas aeruginosa. Antimicrob. Agents Chemother. 28:99-103.
mases remains unresolved. As with the ,B-lactamase studied 9. Livermore, D. M., T. L. Pitt, C. S. Jones, J. A. Crees-Morris,
here, none has been shown to be coded by a transmissible and R. J. Williams. 1985. PSE-4 13-lactamase: a serotype specific
plasmid or by any readily mobilized transposon. On the enzyme in Pseudomonas aeruginosa. J. Med. Microbiol. 19:
other hand, it is clear (except perhaps with the A. hydrophila 45-53.
10. Lugtenberg, B., J. MeUers, R. Peters, P. Van der Hock, and L.
enzymes) that they are not part of the normal enzymatic van Alphen. 1975. Electrophoretic resolution of the "major
complement of these species. Many of these enzymes have outer membrane protein" of Escherichia coli K-12 into four
been described only briefly, and a full comparison of their bands. FEBS Microbiol. Lett. 58:254-258.
properties cannot yet be undertaken. The pI 9.7 enzyme 11. Richmond, M. H., and R. B. Sykes. 1973. The 1-lactamases of
described in this study may or may not be identical to the gram-negative bacteria and their possible physiological role.
enzyme with a pI of ca. 10 observed by Medeiros and Hare Adv. Microb. Physiol. 9:31-88.
(26th ICAAC) in their S. marcescens isolate, crude extracts 12. Roy, C., A. Fox, C. Segura, M. Tirado, C. Foster, and R. Reig.
of which possessed imipenem-hydrolyzing activity. 1983. Plasmid-determined 3-lactamases identified in a group of
It is not known whether most of these uncommon imi- 204 ampicillin-resistant Enterobacteriaceae. J. Antimicrob.
penem-hydrolyzing enzymes are zinc dependent. The en- Chemother. 12:507-510.
13. Saino, Y., F. Kobayashi, M. Inoue, and S. Mitsuhashi. 1982.
zymes described in this study could be inactivated by EDTA Purification and properties of inducible penicillin 13-lactamase
and EGTA, both of which can chelate zinc and other isolated from Pseudomonas maltophilia. Antimicrob. Agents
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vated the pl 9.7 enzyme, and the addition of zinc sulfate to 14. Sato, K., T. Fugli, R. Okamato, M. Inoue, and S. Mltsuhashi.
EDTA- or EGTA-treated enzyme did not restore catalytic 1985. Biochemical properties of ,B-lactamase produced by Fla-
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classify it as a zinc-dependent metalloenzyme. 15. Shannon, K., A. King, and I. Phillips. 1986. 3-Lactamases with
Results of this study, together with those of the studies high activity against imipenem and SCH 34343 from Aeromonas
noted above, make it clear that P-lactamases that are capable hydrophilia. J. Antimicrob. Chemother. 17:45-50.
16. Sirot, D., J. Sirot, R. Labia, A. Morand, P. Courvalin, A.
of hydrolyzing penems and carbapenems occur occasionally Darfeuilie-Michaud, et al. 1987. Transferable resistance to third
in clinically important gram-negative species. However, generation cephalosporins in clinical isolates of Klebsiella pneu-
none of the enzymes reported here seems to be readily moniae: identification of CTX-1, a novel P-lactamase. J. Anti-
transmissible. This is in contrast to the TEM- and SHV- microb. Chemother. 20:323-334.
derived 1-lactamases (e.g., CTX-1, CAZ-1, and SHV-2), 17. Yang, Y., and D. M. Livermore. 1989. Interactions of FCE
which can provide enterobacteria with resistance to broad- 22101 with class I ,B-lactamases. J. Antimicrob. Chemother.
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termined by promiscuous plasmids, which are beginning to 17a.Yang, Y., and D. M. Livermore. 1989. Interactions of mero-
spread between species (6, 16). penem with class I f3-lactamases. J. Antimicrob. Chemother.
24(Suppl. A):207-217.
18. Yang, Y., D. M. Livermore, and R. W. Williams. 1988. Chro-
LITERATURE CITED mosomal P-lactamase expression and antibiotic resistance in
1. Bennett, P. M., J. Heritage, and P. M. Hawkey. 1986. An Enterobacter cloacae. J. Med. Microbiol. 25:227-233.
ultra-rapid method for the study of antibiotic resistance plas- 19. Yotsuji, A., S. Minanis, M. Inoue, and M. Mitsuhashi. 1983.
mids. J. Antimicrob. Chemother. 18:421-424. Properties of novel 3-lactamase produced by Bacteroides fragi-
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