Vol. 35, No. 11

Effect of Clavulanic Acid on Activity of 3-Lactam Antibioticsin Serratia marcescens Isolates Producing Both a TEM

1-Lactamase and a Chromosomal CephalosporinaseKAREN BUSH,t* ROBERT K. FLAMM,t SHARI OHRINGER, SUSAN B. SINGER,

ROSALIE SUMMERILL, AND DANIEL P. BONNERtThe Bristol-Myers Squibb Pharmaceutical Research Institute,

P.O. Box 4000, Princeton, New Jersey 08540

Received 26 March 1991/Accepted 13 August 1991

An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediatedTEM-1 _j-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibilityto extended-spectrum P-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, butit decreased the MICs of the penicillins. The TEM-l-producing plasmid was transferred to a more susceptibleS. marcescens strain that produced a well-characterized inducible chromosomal 13-lactamase. The MICs of theESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin, which were good substratesfor the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were

hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these 3-lactamantibiotics, at least modest increases in chromosomal 3-lactamase activity were observed. When organismswere grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. Theincreases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combinationof low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. Whenclavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a

chromosomal P-lactamase, much higher levels of chromosomal j3-lactamase activity were present than were

observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were

available for induction as a result of inhibition of the TEM enzyme by clavulanate.

Resistance to P-lactam antibiotics is caused to a greatextent by the presence of P-lactamases, enzymes that are

capable of destroying the antibacterial activities of many ofthese agents. Clavulanic acid has become a useful supple-ment to penicillins that are susceptible to hydrolysis byplasmid-mediated P-lactamases. This 3-lactamase inactiva-tor works well when it is combined with amoxicillin orticarcillin to treat infections caused by bacteria that producemany kinds of penicillinases or P-lactamases with broad-spectrum hydrolyzing activities (1, 17). However, recentreports indicate that occasional antagonism of penicillinactivity has been observed when antibiotic combinationswith clavulanic acid are added to selected gram-negativerods (19, 21). In certain isolates, the lack of synergy ob-served has been attributed to the induction of chromosomalcephalosporinases (10, 12, 22), P-lactamases that are notwell inhibited by clavulanate. Although most studies haveincluded organisms that produce only a single induciblechromosomal cephalosporinase, some resistant isolates havebeen identified as strains that produce multiple P-lactamases(8, 19).

In our studies, we have observed a small population ofgram-negative organisms with decreased susceptibilities toextended-spectrum ,-lactam antibiotics (ESBAs). In theseisolates, the microbiological activities of ESBAs were notincreased by the addition of clavulanic acid, but the activi-ties of penicillins were improved. Many of these organisms

* Corresponding author.t Present address: Medical Research Division, American Cyana-

mid Co., Pearl River, NY 10965.t Present address: Bristol-Myers Squibb, Wallingford, CT 06492.

were shown to produce an inducible chromosomal cephalo-sporinase in addition to a plasmid-mediated P-lactamase. Inthis report we describe the characterization of one of theseorganisms, a clinical isolate of Serratia marcescens, that wasoriginally identified in a group of aminoglycoside-resistantorganisms (6). The P-lactamase-producing plasmid activitywas transferred to another prototype strain of S. marcescensto determine whether the anomalous antibiotic -activitieswere plasmid associated. Biochemical characterization ofthe ,-lactamases and outer membrane proteins (OMPs)allowed proposal of a mechanism (2) that encompassed a

combination of effects resulting in the observed microbio-logical activities.

MATERIALS AND METHODS

Chemicals. Amoxicillin, ampicillin, and aztreonam were

from Bristol-Myers Squibb (Princeton, N.J.). Cephaloridinewas from Eli Lilly (Indianapolis, Ind.). Cefotaxime was

obtained from Hoechst-Roussel (Raritan, N.J.). Ceftriaxonewas from Roche (Nutley, N.J.). Clavulanic acid was fromBeecham (Knoxville, Tenn.). Phenylmethylsulfonyl fluo-ride, Sarkosyl (N-lauroylsarcosine), and 2-mercaptoethanolwere from Sigma Chemical Co. (St. Louis, Mo.). ProBluestain was from ISS Enprotech (Hyde Park, Mass.). Sodiumdodecyl sulfate, bisacrylamide, and acrylamide were fromBio-Rad (Richmond, Calif.).

Organisms. S. marcescens SC 15071, which producescephalosporinase S1 and the plasmid-mediated TEM-1 P-lac-tamase (plasmid pSVA071), was from a group of clinicalisolates selected at the Seattle Veterans Affairs MedicalCenter on the basis of resistance to gentamicin (6). S.

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marcescens SC 9782, which produces cephalosporinase S2,was a clinical isolate that was resistant to penicillins. Esch-erichia coli K-12 (SC 12759) was used as the recipientorganism for the production of transconjugants. E. coli SC15111 was a transconjugant obtained from S. marcescens SC15071 and E. coli SC 12759. S. marcescens SC 15110 was atransconjugant obtained from E. coli SC 15111 and S.marcescens SC 9782.

Transconjugants. Transconjugants were generated by mix-ing donor and recipient organisms on diagnostic sensitivitytest agar. After overnight incubation at 37°C, growth wasscraped from the agar and plated onto the appropriateselective medium, as follows: for E. coli SC 15111, ampicillinat 100 ,ug/ml and nalidixic acid at 100 ,ug/ml; for S. marces-cens SC 15110, ampicillin at 50 ,ug/ml on Simmons citrateagar. Plasmid isolation procedures (14) confirmed plasmidtransfer.

Microbiological assays. MICs were determined by agardilution assays on diagnostic sensitivity test agar (13, 20).

P-Lactamase induction. Cultures producing ,-lactamaseswere grown overnight and were inoculated into modifiedDavis medium. After transfer of cells to give a 2% inoculum,organisms were grown for 2.0 h. Inducer or phosphate buffer(in duplicate for uninduced controls) was then added 1.0 hbefore harvest. Cells were washed and sonicated in 0.05 Mphosphate buffer (pH 7.0) (3). Immediately following centrif-ugation, clarified cell extracts were assayed for P-lactamaseactivity by using the chromogenic cephalosporin SQ 24902.Assays were performed spectrophotometrically on a Gilford250 spectrophotometer at 25°C in 0.05 M phosphate buffer(pH 7.0). The total amount of P-lactamase was calculated foreach sample and was compared with the amount for theuninduced controls run on the same day.Sl P-lactamase purification. For kinetic studies, the S1

chromosomal P-lactamase was separated from the TEM-1,B-lactamase by QAE-Sephadex chromatography (18). S.marcescens SC 15071 was grown and harvested as describedabove, with maximal SI enzyme production ensured byinduction with ampicillin (100 tg/ml).' Cells were broken bya freeze-thaw procedure (3). Protein was precipitated bybringing the solution to 95% saturation with ammoniumsulfate. The resulting precipitate was frozen overnight be-fore suspension and dialysis in 0.025 M TES buffer (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) (pH8.0) and was eluted from QAE-Sephadex with the samebuffer (TES). S1 P-lactamase appeared immediately in theeffluent, while the TEM-1 activity was eluted in later frac-tions. The identities and purities of all P-lactamases wereevaluated by isoelectric focusing followed by staining for1-lactamase activity with SQ 24902 (PAGplates [pH range,3.5 to 9.5] by using an LKB Multiphor electrophoresis unit.)

Kinetic -determinations. Hydrolysis studies were per-formed spectrophotometrically as described previously (15).All assays were conducted by using preparations containingthe activity of a single enzyme. The TEM-1 P-lactamase wasfrom an uninduced E. coli SC 15111 preparation, and the S2enzyme was from an amoxicillin-induced S. marcescens SC9782 extract. Kinetic parameters (Vmax and Kin) were deter-mined by using the program ENZPACK (Elsevier), whichcalculates kinetic parameters by four different methods.Inhibition studies were performed by incubating clavulanicacid with purified enzyme for 5 min before the addition of 1.0mM cephaloridine. The A295 was monitored for at least 5min. Initial hydrolysis rates were used for the determina-tions.OMPs. OMPs were prepared as described by Williams

(23), with the following modifications. Organisms were inoc-ulated from overnight cultures grown in Mueller-Hintonbroth at 37°C. After 3 h, cells were incubated with antibioticat the MIC in the presence or absence of clavulanic acid andwere harvested after 1 h of growth. After two washes with 10mM phosphate buffer (pH 8.0), the cells were suspended inthe same buffer containing phenylmethylsulfonyl fluoride.Disrupted cells were washed twice with the phenylmethyl-sulfonyl fluoride buffer. Following solubilization in Sarkosyl,membranes were centrifuged for 30 min at 10°C. Pellets weredissolved in the buffer described by Laemmli (9) withoutmercaptoethanol, and protein concentrations were deter-mined by the Pierce Protein Assay (Pierce Chemical Com-pany, Rockford, Ill.). Approximately 30 ,ug of protein wasapplied to the 12% polyacrylamide gel polymerized in theabsence of sodium dodecyl sulfate. However, sodium dode-cyl sulfate was included in the running buffer. The gel wasrun for 22 h at room temperature before it was stained withProBlue.

RESULTS

Generation of transconjugants. S. marcescens SC 15071was a multiresistant clinical isolate that contained a singleplasmid. Plasmid pSVA071 was transferred sequentially toE. coli K-12 and then to S. marcescens SC 9782, to give thetransconjugants W. coli SC 15111 and S. marcescens SC15110, respectively. No other plasmids were observed in anyof the organisms in this study.

Identities of P-lactamases. The two clinical isolates, SC9782 and SC 15071, produced nonidentical P-lactamases withisoelectric points of 7.1 and >9.0, respectively. Both ,-lac-tamase activities were increased when the organisms weregrown in the presence of cefoxitin. Clinical isolate SC 15071also produced a second ,B-lactamase, an enzyme with a pI of5.4, corresponding to that of TEM-1. This TEM band wa'sidentical to that observed in extracts from the transconju-gants E. coli SC 15111 and S. marcescens SC 15110, indi-cating the presence of the plasmid-mediated enzyme'in eachconstruction.

Microbiological activity. The MICs of selected penicillinsand ESBAs were determined in the presence and absence ofclavulanic acid, as shown in Table 1. In the absence ofclavulanic acid, MICs of penicillins were >800 ,ug/ml forthose organisms that produced a plasmid-mediated TEM,B-lactamase. However, for strains SC 15110 and SC 15111,the MICs of the ESBAs were not elevated above those forthe isogenic wild-type organisms (SC 9782 and SC 12759),except for the MIC of ceftriaxone for S. marcescens SC15110. MICs of all P-lactam antibiotics were elevated for theTEM-producing strain S. marcescens SC 15071, in compar-ison with those for SC 9782, both in the presence andabsence of clavulanic acid.The addition of clavulanic acid lowered the MICs of the

penicillins for all the TEM producers. However, for SC15071, again,'the MICs remained very high. The addition ofclavulanic acid to the ESBAs resulted in no change in theMICs for the two strains of E. coli, regardless of enzymeproduction. For the S. marcescens strains SC 9782 and itsderivative SC 15110,' clavulanic acid had no effect on theactivity of aztreonam and served to increase the MICs ofcefotaxime and ceftriaxone by 1 or 2 dilutions. All MICs forthese strains remained below 1 pugIml. The addition ofclavulanic acid to extended-spectrum antibiotics increasedall MICs two- to fourfold for S. marcescens SC 15071,

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TABLE 1. Effect of clavulanic acid on microbiological activity

*CA concn MIC (pg/ml)bOrganism (IWg/ml) Ampicillin Amoxicillin Cefotaxime Ceftriaxone Aztreonam

S. marcescens SC 15071(pSVA071) 0 >800 >800 6.3 3.1 0.82 400 400 12.5 6.3 3.1

S. marcescens SC 9782 0 50 100 0.4 0.2 0.22 50 100 0.8 0.4 0.2

S. marcescens SC 15110(pSVA071) 0 >800 >800 0.2 0.8 0.22 50 100 0.8 0.8 0.2

E. coli SC 12759 0 6.3 12.5 0.1 <0.05 0.12 3.1 12.5 0.1 <0.05 0.1

E. coli SC 15111(pSVA071) 0 >800 >800 0.1 <0.05 0.12 12.5 25 0.1 <0.05 0.1

a CA, clavulanic acid.b Agar dilution assays with inoculum of 106 CFU.

resulting in MICs 4 to 16 times higher than those for of theprototype strain S. marcescens SC 9782.

j-Lactamase production. To determine whether the differ-ences in microbiological activities between SC 15071 and theother S. marcescens strains were due simply to the amountof enzyme produced, the total P-lactamase activity wasdetermined in crude sonic extracts of each of the organisms.The results are given in Table 2. In uninduced cultures, SC15071 produced approximately four times the basal level ofchromosomal P-lactamase activity observed in S. marces-cens SC 9782. The level ofTEM activity was similar in the E.coli SC 15111 and S. marcescens strains.

P-Lactamase kinetic properties. As shown in Table 3, theS1 and S2 enzymes did not hydrolyze ampicillin or amoxi-cillin well and exhibited poor hydrolysis of the ESBAs. S1cephalosporinase had higher Vmax values for the ESBAsthan did the S2 enzyme. However, when VmaxIKm valueswere compared for the ESBAs, there were no major differ-ences between the S1 and S2 enzymes. As expected, thepenicillins were excellent substrates for the TEM-1 enzyme,but the ESBAs were only poorly hydrolyzed. The TEMenzyme was well inhibited by clavulanic acid, with a 50%inhibitory concentration of 0.23 ,uM (0.055 ,ug/ml). The Siand S2 enzymes were only weakly inhibited, with 50%

TABLE 2. Production of ,B-lactamases

Total 1B- CalculatedCulture lactamase Enzyme enzyme

activitya activity

S. marcescens SC 15071(pSVA071) 763 S1 273TEM-1 490b

S. marcescens SC 9782 64 S2 64S. marcescens SC 15110(pSVA071) 554 S2 64c

TEM-1 490E. coli SC 12759 <15 (AmpC) <15E. coli SC 15111(pSVA071) 659 (AmpC) <15d

TEM-1 644a Nanomoles of cephaloridine hydrolyzed per minute per milligram of

protein in crude extract.b Assumed to be equal to TEM activity in SC 15110.c Assumed to be equal to S2 activity in SC 9782.d Assumed to be equal to AmpC activity in SC 12759.

inhibitory concentrations of 70 ,uM (17 ,ug/ml) and 93 puM (22,ug/ml), respectively.

Induction of I-lactamases. The influence of clavulanic acidon the production of P-lactamase activity for each S. mar-cescens strain was determined for each of the antibiotics. Asshown in Table 4, ampicillin and amoxicillin were veryeffective inducers of the S2 1-lactamase in SC 9782, but theyexhibited induction differentials of only 1.5 and 1.1, respec-tively, in the presence of clavulanic acid. The extended-spectrum antibiotics were not strong inducers of the S2,B-lactamase. Clavulanic acid alone did not appear to be aneffective inducer of the S2 1-lactamase when it was tested at2 p.g/ml in strain SC 9782.A more complex situation presented itself in the induction

data for those organisms that produced multiple P-lactam-ases. The results in Table 4 indicate the calculated values forthe contribution of each enzyme to the total ,B-lactamaseactivity. Two assumptions were made to obtain these re-sults. First, the basal level of S2 enzyme was not affected bythe presence of plasmid pSVA071, and second, the level ofTEM ,B-lactamase would be essentially constant in both S.marcescens strains.

In the absence of clavulanic acid, the induction effect fromthe penicillins in strain SC 15110 was less than that observedin the isogenic strain SC 9782, which contains the activity ofa single ,-lactamase. However, when subjected to treatmentwith clavulanic acid in the presence of either ampicillin oramoxicillin, both strains of S. marcescens containing thepSVA071 plasmid exhibited large increases in P-lactamaseactivity compared with the activity following induction withpenicillin alone. Note, in particular, this increase in strainSC 15110, both with respect to induction by the penicillinalone and with respect to strain SC 9782 in the presence ofclavulanate and a penicillin. This behavior was not observedwith the extended-spectrum antibiotics. With the addition ofclavulanic acid, there was no increase in 13-lactamase activ-ity compared with that after induction by the ESBA alone.OMP profiles. Examination of the OMP profiles revealed

that the two strains differed in several areas (Fig. 1). StrainSC 15071 exhibited a strong single OMP band located at 40kDa, which is in contrast to the results for strains SC 9782and SC 15110, which had major doublet bands at 41 and 40kDa. Strain SC 15071 also had a weak doublet in the 37- to

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TABLE 3. Kinetic parameters of 1-lactamases

Si S2 TEM-1Substrate Km Relative Relative Km Relative Relative Km Relative Relative

(>LM) Vmax Vmax/Km (>tM) Vmax VmKm (>LM) Vmax Vmax/Km

Cephaloridine 470 100 100 400 100 100 340 100 100Ampicillin, 1600 NCa <0.1 NC 290 0.034 0.047 16Amoxicillin NC <0.3 NC NC <0.05 NC 77 170 750Cefotaxime 240 0.26 0.51 31 0.054 0.70 NC <0.005 NCCeftriaxone 46 0.23 2.4 15 0.035 0.93 34 0.033 0.33Aztreonam 160 0.058 0.17 33 0.041 0.50 220 0.30 0.46

a NC, not calculated. Rates were too slow to determine Km reliably.

38-kDa region and a strong doublet in the 36-kDa region. clavulanic acid inhibited the plasmid-mediated TEM ,-lac-Strains SC 9782 and SC 15110 exhibited no bands in the 37- tamase, an enzyme that is readily inactivated by concentra-to 38-kDa area and only a singlet in the 36-kDa region. Each tions much lower than 2 ,ug of clavulanate per ml. In strainsstrain exhibited the same qualitative banding patterns as that produced a TEM enzyme, this broad-spectrum enzymedescribed above, when cells were grown in either clavu- would easily hydrolyze the penicillins ampicillin or amoxi-lanate or aztreonam, or both (data not shown). cillin if no inhibitor were present. Thus, the concentration of

penicillin available for induction of the chromosomal en-DISCUSSION zyme would be much lower in the absence of clavulanate

compared with that in an isolate that produces only aDecreased susceptibilities to ESBAs in the presence of chromosomal enzyme. This explanation is consistent with

clavulanic acid have been reported in various gram-negative the data in Table 4, which show that both the Si and S2organisms (4, 19, 21) and have been attributed by some ,B-lactamases in the multiple enzyme producers were in-investigators to the induction of group I -lactamases by duced at lower relative levels compared with the levelsclavulanate, a phenomenon that has been observed in se- needed for induction of the S2 enzyme by penicillins alone inlected strains under various conditions (5, 11, 12). Often, strain SC 9782. In the latter organism, therefore, there musthowever, organisms producing only a chromosomal 1-lacta- be more intact penicillin available for induction.mase were examined. The effect of clavulanic acid in com- In the S.tacescen clal for SC10 sebination with a second ,B-lactam antibiotic has not been In the S. marcescens clinical isolate SC 15071, severalevaluated biochemically in strains that produce multiple factors could be identified as contributing to the decreasedP-lactamases, an increasingly common phenomenon. susceptibility of ESBAs in combination with clavulanic acid.When clavulanic acid was combined with ampicillin or Because both SC 15071 and SC 15110 had similar qualitative

amoxicillin in the S. marcescens strains that produced both induction effects, induction by clavulanate did not play aa TEM-1 enzyme and a chromosomal cephalosporinase, major role in causing decreased susceptibility. This obser-total 1-lactamase production was quite high compared with vation is consistent with the studies of Livermore et al. (10)the activity in strains induced only with penicillins. This in which introduction of a TEM-1 ,B-lactamase into Entero-should not be interpreted to mean that clavulanic acid was bacter cloacae had little or no effect upon induction byacting like a strong inducer. If this were the case, the same cefotaxime-clavulanate. Low-level induction of the chromo-effect should have been observed in S. marcescens SC 9782, somal enzymes by the ESBAs alone or in combination witha strain in which clavulanic acid alone did not cause an clavulanate occurred in all S. marcescens strains. With evenincrease in P-lactamase activity. It is most probable that small increases in P-lactamase levels, additional factors such

TABLE 4. P-Lactamase activities calculated with respect to individual enzyme contributions

OrganismEnzymeCA concn ,B-Lactamase activities calculated after inductionbOrganism Enzyme C oc

(,Wg/ml)' Control Ampicillin Amoxicillin Cefotaxime Ceftriaxone Aztreonam

S. marcescens SC 15071 Si 0 2.7 (1.0) 32 (12) 24 (8.9) 15 (5.4) 8.5 (3.1) 11 (4.0)TEM 0 3.1 (1.0) 3.1 (1.0) 3.1 (1.0) 3.1 (1.0) 3.1 (1.0) 3.1 (1.0)S1 2 1.2 (0.44) 195 (72) 300 (110) 9.3 (3.4) 7.5 (2.8) 9.3 (3.4)TEM 2 1.1 (0.36) 1.1 (0.36) 1.1 (0.36) 1.1 (0.36) 1.1 (0.36) 1.1 (0.36)

S. marcescens SC 9782 S2 0 0.42 (1.0) 19 (46) 28 (66) 0.55 (1.3) 1.1 (2.7) 0.50 (1.2)S2 2 0.50 (1.2) 28 (67) 30 (72) 0.63 (1.5) 1.3 (3.2) 0.63 (1.5)

S. marcescens SC 15110 S2 0 0.42 (1.0) 9.8 (23) 9.8 (23) 2.5 (6.0) 11 (25) 3.9 (9.3)TEM 0 3.1 (1.0) 3.1 (1.0) 3.1 (1.0) 3.1 (1.0) 3.1 (1.0) 3.1 (1.0)S2 2 0.50 (1.2) 65 (150) 55 (130) 1.4 (3.4) 10 (24) 3.1 (7.3)TEM 2 1.1 (0.36) 1.1 (0.36) 1.1 (0.36) 1.1 (0.36) 1.1 (0.36) 1.1 (0.36)

a CA, clavulanic acid.b Induction of ,B-lactamases was performed in the absence or presence of clavulanic acid by using ampicillin or amoxicillin at 100 ,ug/ml; cefotaxime,

ceftriaxone, and aztreonam were used at the MICs. Activity is nanomoles of the cephalosporin SQ 24,902 hydrolyzed per minute per milligram of cells. Valuesin parentheses are normalized values in comparison with values for controls not treated with antibiotic or clavulanic acid.

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-43 kD

-31 kD

X -21.5 kD

a b c 4

FIG. 1. OMPs of S. marcescens. Lane a, SC 15071; lane b, SC9782; lane c, SC 15110; lane d, molecular mass markers.

as hydrolysis differentials and permeability become impor-tant.

Identification of causative factors other than induction isconsistent with the observations of other investigators (5,16). Certainly, differences in the hydrolysis of ESBAs by S1and S2 ,-lactamases must be evaluated, considering the factthat the S1 enzyme was produced at a higher basal level thanthe S2 enzyme was. However, even when the S2 enzymewas induced to levels that approached or exceeded the basallevel of enzyme in SC 15071, the MICs of all antibiotics forSC 15110 were virtually identical to those for its parent anddid not increase to the levels observed for SC 15071. Theincreased hydrolysis of ceftriaxone by the S1 enzyme com-pared with that by the S2 enzyme may have been significantwhen small increases in ,B-lactamase concentrations oc-curred. It is clear, however, that introduction of plasmidpSVA071 did not affect the permeability or the susceptibilityof SC 9782 or SC 15110 to the ESBAs or the penicillin-clavulanate combinations.The differences in OMP profiles between the two strains of

S. marcescens may also be important, although it has notbeen established that the observed changes are related topermeability. The OMP patterns of SC 9782 and SC 15110were similar to that described by Gutmann et al. (7) for awild-type S. marcescens strain. In that study, a mutantselected on the basis of resistance to nalidixic acid, trimeth-oprim, and chloramphenicol exhibited a decrease in thequantity of the 41-kDa protein and an increase in the amountof the 40-kDa protein, a pattern similar to that of strain SC15071. Thus, OMP differences could be related to the de-creased susceptibility of strain SC 15071 compared with thatof strain SC 9782 or SC 15110. This is consistent with thesuggestion of Thomson et al. (21) that the decreased poten-cies of P-lactam combinations containing clavulanate mightbe related to outer membrane permeability.As emphasized in this study, susceptibility to P-lactam

antibiotics is dependent on several interrelated factors.When a TEM-producing plasmid was introduced into asecond strain of S. marcescens, the plasmid did not conferthe parental antibiotic phenotype to the recipient strain. Infact, quite different microbiological activities were observedbetween the two strains that produced multiple P-lactam-ases. Induction phenomena were shown to be dependent onthe characteristics of the various enzymes present, withcompetition among substrates, inducers, or inhibitors being

an important consideration. When combinations of ,-lactamantibiotics are used clinically, it may be very difficult tomake accurate predictions about the success of this therapybecause of the interplay of a variety of interactions, espe-cially in organisms that produce multiple enzymes.

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