ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1994, p. 767-772 Vol. 38, No. 40066-4804/94/$04.00+0Copyright 1994, American Society for Microbiology

Comparative Activities of Clavulanic Acid, Sulbactam, andTazobactam against Clinically Important 3-Lactamases

DAVID J. PAYNE, REBECCA CRAMP, DAVID J. WINSTANLEY, AND DAVID J. C. KNOWLES*SmithKline Beecham Pharmaceuticals, Brockham Park, Betchworth, Surrey RH3 7AJ, United Kingdom

Received 16 April 1993/Returned for modification 14 July 1993/Accepted 26 January 1994

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated Il-lactamases.However, inhibition data for these three inhibitors with a wide range of different plasmid-mediatedP-lactamases have not yet been compared under the same experimental conditions. A number of groups haveinferred that clavulanic acid inhibits extended-spectrum TEM and SHV P-lactamases, but inhibition data haverarely been published. In this study, the 50%o inhibitory concentrations of these three ,-lactamase inhibitorsfor 35 plasmid-mediated P-lactamases have been determined. Of these 35 P-lactamases, 20 were extended-spectrum TEM- or SHV-derived I-lactamases. The other 15 enzymes were conventional-spectrum P-lacta-mases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the35 plasmid-mediated I-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent thansulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negativeplasmid-mediated I-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showedthat clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrumenzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the twogroups of P-lactamases. There were no significant differences between the overall activities of tazobactam andclavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes,although differences in their inhibition profiles were observed.

I-lactamases are plasmid-encoded or chromosomally en-coded bacterial enzymes which hydrolyze 3-lactam antibiotics.Plasmid-mediated ,-lactamases can transfer rapidly betweenbacterial genera and consequently pose a major threat to thesuccessful use of ,-lactam agents. More than 60 different typesof plasmid-encoded ,-lactamases have been characterized,and for the purpose of this work, the enzymes have beenclassified as either conventional-spectrum or TEM- and SHV-derived extended-spectrum enzymes. The conventional-spec-trum ,-lactamases are from Bush groups 2a, 2b, 2c, and 2d,and extended-spectrum TEM and SHV enzymes are all fromgroup 2b' (4).The conventional-spectrum enzymes include enzymes such

as TEM-1 and SHV-1, which do not confer resistance tocephalosporins such as ceftazidime and cefotaxime. A recentsurvey of 802 gram-negative clinical isolates showed thatTEM-1 and SHV-1 were responsible for mediating P-lactamresistance in 17% of clinical isolates (37). Other conventional-spectrum enzymes which are often found in clinical isolatesinclude the OXA and PSE P-lactamases (1). The most com-mon conventional-spectrum plasmid-mediated 13-lactamasefound in gram-positive bacteria is the penicillinase producedby the majority of Staphylococcus aureus clinical isolates (26).Most of the extended-spectrum plasmid-mediated ,B-lacta-

mases are derived from the TEM and SHV ,B-lactamase genesand confer transferable resistance to the newer broad-spec-trum cephalosporins such as cefotaxime and ceftazidime. Sofar, at least 25 to 30 different extended-spectrum ,-lactamaseshave been reported, and none of the enzymes appear todominate the clinical situation worldwide. Therefore, a broad

* Corresponding author. Mailing address: SmithKline BeechamPharmaceuticals, Brockham Park, Betchworth, Surrey RH3 7AJ,United Kingdom. Phone: 737 364518. Fax: 737 364597.

selection of extended-spectrum 1-lactamases must be exam-ined when assessing the activities of antimicrobial agentsagainst such enzymes. The clinical prevalence of extended-spectrum ,B-lactamases in certain areas is increasing (35) andconsequently compromises the effectiveness of the newercephalosporins (17).

Combinations of ,B-lactam antibiotics and ,-lactamase inhib-itors have proved successful at treating infections caused bybacteria producing 13-lactamases (22). The potency of suchcombinations continues to be largely assessed by susceptibilitytesting (2). Such studies have suggested that clavulanic acidand other P-lactamase inhibitors differentially inhibit varioustypes of plasmid-mediated 3-lactamases (15, 16, 19). However,the activity of a particular combination against clinical isolatesis affected by the interplay of many different factors which arediscussed in detail by Thomson et al. (36). One major compo-nent which affects the overall antibacterial activity of a ,-lac-tam-3-lactamase inhibitor combination is the susceptibility ofthe 3-lactamase to the inhibitor, and it is this component whichhas been addressed in our study. As there are now manydifferent types of plasmid-mediated 3-lactamase, any assess-ment of the potency of different ,B-lactamase inhibitors must beperformed on a broad range of 3-lactamases. Therefore, thispaper reports the results of a comprehensive study which hasexamined the ,B-lactamase inhibitory activities of clavulanicacid, sulbactam, and tazobactam against 35 isolated plasmid-mediated ,-lactamases. Twenty of the enzymes were extended-spectrum ,B-lactamases, and the remainder were conventional-spectrum plasmid-mediated ,B-lactamases.

MATERIALS AND METHODS

Antibiotics. Sulbactam was supplied by Pfizer Central Re-search, and tazobactam was obtained from Lederle Laborato-ries. Clavulanic acid was prepared in our laboratories.

767

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Strains and plasmids used in studyBush group and strain Plasmida P-Lactamase Reference

Group 2a ,-lactamasesS. aureus Russell Staphylococcal penicillinase

Group 2b j3-lactamasesE. coli K-12 J53-2 Pro- Met- Rf'

Group 2b' l-lactamasesE. coli C600 Nalr The - Leu- Thi -

E. coli K-12 J53-2 Pro- Met- Rf'E. coli C600 Nalr The- Leu- Thi -E. coli K-12 J53-2 Pro- Met- Rf'

E. coli BM 694 Nalr, Cla derivedE. coli K-12 J53-2 Pro- Met- Rf

Klebsiella oxytoca

Group 2c P-lactamasesPseudomonas aeruginosa PU21 liv-

Leu-. Strr RfBranhamella catarrhalis

Group 2d 3-lactamasesE. coli K-12 J53-2 Pro- Met- Rf'

P. aeruginosa PA038 Leu- Rf NalrP. aeruginosa PU21 lv- Leu- Strr Rf'

R6KRP4R1010

pCF04pCFF14pMG226pLF100pMG228pJPQ100pUD17pUD18pAFF1100Kb100KbRP4CRP4DR1818EpUK700pUK721pUK723pUK724pCFF44pUK730pMG204b

14

TEM-1TEM-2SHV-1

131330

TEM-3TEM-5TEM-6TEM-7TEM-9TEM-10SHV-2SHV-3SHV-SEnzyme AEnzyme BEnzyme CEnzyme DEnzyme ETEM-ElTEM-E2TEM-E3TEM-E4CAZ-3DJP-1TLE-1MJ-1

3329312343120181127b27b27b27b27b27b27b27b27b3227b258

PSE-4

BRO-1

R455R46pMG203pMG54pMG204bpMG39R151

10

9

OXA-1OXA-2OXA-4OXA-5OXA-7OXA-6PSE-2

772525252523

a The plasmid is identified if it was named in the original publication.b Including references cited within.

Strains and plasmids. The Escherichia coli transconjugantstrains used in this study are listed in Table 1. These strainswere selected because they produced high levels of plasmid-mediated P-lactamase. Enzymes A and B were obtained fromTEM-1 by in vitro spontaneous mutation; in the same way,enzymes C and D were derived from TEM-2 and enzyme E wasderived from PSE-4 (27). TEM-E2, TEM-El, CAZ-lo, andTEM-E4 have pIs similar to those of enzymes A, B, C, and D,respectively (27). However, isoelectric focusing data in isola-tion are not sufficient to assess whether two TEM ,-lactamaseswith similar pIs are identical (17). The S. aureus RussellP-lactamase was representative of a typical staphylococcalpenicillinase.

Preparation of I8-lactamases. A 1-liter culture (nutrientbroth no. 2) of each strain listed in Table 1 was shakenovernight at 37C. The cells were harvested by centrifugationfor 15 min at 6,000 x g. The bacterial pellets were washed in

25 mM sodium phosphate buffer (pH 7), and the centrifugationwas repeated. One milliliter of 25 mM sodium phosphatebuffer was then added to the final pellet, and the cells wereresuspended to give 3 ml of cell suspension, which wasdisrupted by ultrasound. The cell lysate was cleared by centrif-ugation for 1 h at 32,000 x g.

,-Lactamase identification. Each 13-lactamase was checkedby analytical isoelectric focusing (24). The enzymes wereexamined on gels containing a 1:1 ratio of pH 3.5 to pH 10ampholines and either pH 4 to pH 6 or pH 9 to pH 11ampholines, depending on the expected pI of the P-lactamase.The prepared enzymes were focused alongside known 3-lacta-mase preparations. Each preparation from the E. coli transcon-jugants was shown to produce only one plasmid-mediatedenzyme with a trace amount of the E. coli K-12 chromosomalenzyme. The other strains all produced the single plasmid-mediated enzyme.

768 PAYNE ET AL.

COMPARATIVE ACTIVITIES OF INHIBITORS OF ,B-LACTAMASES 769

TABLE 2. Geometric means and 95% confidence intervals forIC50s of clavulanic acid and tazobactam for OXA-1 and SHV-5

IC50 (PM),B-lactamase and No. of deter-

inhibitor minations Range Geometric 95% Ciamean

OXA-1Clavulanic acid 8 1.19-2.11 1.58 1.34-1.84Tazobactam 8 1.00-1.98 1.45 1.21-1.75

SHV-5Clavulanic acid 9 0.01-0.02 0.013 0.009-0.016Tazobactam 9 0.06-0.10 0.076 0.066-0.086a 95% CI, 95% confidence interval.

Assay conditions for the determination of IC50s. The activ-ities of 3-lactamase inhibitors were assessed by determiningthe concentration of inhibitor which inhibits by 50% thehydrolysis of nitrocefin by a particular P-lactamase (IC50).The IC50s were determined by an automated microtiter

assay system as described previously (28). The amount ofenzyme was normalized to give approximately 70 ,uM nitroce-fin hydrolyzed per min.

Inhibitors were assayed at eight different concentrations,ranging from 0.005 to 100 ,uM. The dilution of each inhibitorand its subsequent transfer into microtiter plates were per-formed automatically with a Hamilton Micro Lab AT Plus.The 1-lactamase and inhibitor were preincubated for 5 min at37C, and the assay was initiated by the rapid addition ofnitrocefin to create a final concentration of 0.2 mM.

Calculation of IC50s. The KinetiCalc software provided withthe Biotek reader allowed the plots of absorbance against timefor all 96 wells to be simultaneously displayed in real time. Theinitial rates of hydrolysis at each inhibitor concentration werecalculated, and the IC50s (,IM) were determined by plottingpercentage inhibition against inhibitor concentration (28).

Statistical analysis of IC50 data. The reproducibility of themethodology was examined by measuring the IC50s of clavu-lanic acid and tazobactam for SHV-5 and OXA-1 on at leasteight separate occasions. Analyses were performed on theIC50s after log1o transformation, and geometric means with95% confidence intervals were calculated for each inhibitorwith each enzyme. The reproducibility was assessed by express-

10090

c7080o 70

._

M 40-

302010

-I

0

/ ,A

/,

's

A.,

0.001 0.01 0.1 1 10 100

Concentration of Inhibitor (iM)FIG. 1. Inhibition of S. aureus (Russell) penicillinase by clavulanic

acid (0), sulbactam (A), and tazobactam (-).

TABLE 3. IC50s of P-lactamase inhibitors for 35 plasmid-mediated 13-lactamases

ICso (>.M)P-lactamase

Clavulanic acid Sulbactam Tazobactam

S. aureus Russell 0.28 26 2.3TEM-1 0.09 6.1 0.04TEM-2 0.18 8.7 0.05SHV-i 0.03 17 0.14TEM-3 0.03 0.03 0.01TEM-5 0.03 1.2 0.28TEM-6 0.12 0.45 0.17TEM-7 0.10 0.62 0.18TEM-9 0.29 0.90 0.34TEM-10 0.03 0.34 0.08SHV-2 0.05 2.8 0.13SHV-3 0.04 2.7 0.10SHV-5 0.01 0.63 0.08Enzyme A 0.09 0.48 0.07Enzyme B 0.01 0.12 0.01Enzyme C 0.33 10 0.09Enzyme D 0.04 0.57 0.01Enzyme E 0.09 3.6 0.11TEM-El 0.05 0.64 0.02TEM-E2 0.09 1.6 0.05TEM-E3 0.02 0.20 0.06TEM-E4 0.06 0.79 0.04CAZ-3 0.13 2.5 0.06DJP-1 0.01 0.21 0.02TLE-1 0.11 5.5 0.05MJ-1 0.09 40 0.43PSE-4 0.15 3.7 0.10BRO-1 0.02 0.02 0.02OXA-1 1.8 4.7 1.4OXA-2 1.4 0.14 0.01OXA-4 8.4 16 5.6OXA-5 3.1 18 0.25OXA-6 1.6 51 1.7OXA-7 0.36 40 0.61PSE-2 0.81 37 0.94

ing the range of the confidence interval as a percentage of themean IC50.

Statistical analysis of the IC50s after log1o transformationwas performed by analysis of variance, taking into account theinhibitor, the enzyme, and the two groups of enzymes (con-ventional-spectrum and extended-spectrum TEM and SHV).Differences in the geometric means were back-transformed toratios, i.e., relative potencies, of the inhibitors for the differentgroups of enzymes. The corresponding confidence limits werealso determined.

RESULTS

Table 2 shows the statistical analysis of at least eight IC50sfor four different enzyme-inhibitor interactions. This illustratesthat the semiautomated technique described in this paper wasacceptably reproducible, with 95% confidence intervals lessthan 40% of the geometric mean in three of the four enzyme-inhibitor interactions. The 95% confidence interval for thefourth combination (clavulanic acid with SHV-5) was within60% of the geometric mean (Table 2).

Figure 1 shows the inhibition profiles from which the IC50sfor S. aureus Russell 13-lactamase were calculated. This illus-trates that clavulanic acid was 93 times more active thansulbactam and 8 times more active than tazobactam against theS. aureus enzyme. The graphs for other IC50s are not shown,

VOL. 38, 1994

ANTiMICROB. AGENTS CHEMOTHER.

a0.01 0.1

I50 (MM)1 10 100

b I50 (jIM)0.01 0.1 1 10 100I I~~~~~~~~~~~~~~~

o BRO-1

---------------------0 SHV-1* ----------------0 TEM-1*------------------------OMJ-1

*------------- -O TLE-1*------------0 PSE-4*--------------- TEM-2

------------------O RUSSELLv -------- - o OXA-7

-------------- o PSE-2-------------- OXA-6---- 0 OXA-1

*-------o OXA-5* - -0 OXA-4

OXA-2 0-0-------0

* BRO-1*- - --- 0 SHV-1

0----- MJ-10-------0 RUSSELL

0 0 OXA-7

o PSE-2

0 OXA-6

TEM-1 - -.

TLE-1 0- - 4

PSE-4TEM-2 -----

OXA-2 0 ------------- --

OXA-1 OXA-5 0-------- 0

OXA-4 0-FIG. 2. Comparison of the IC50s of 3-lactamase inhibitors against conventional-spectrum P-lactamases. (a) IC50s of clavulanic acid (0) and

sulbactam (0); (b) IC50s of clavulanic acid (0) and tazobactam (0).

and subsequent discussion of inhibition data refers directly toIC50s (these are tabulated in Table 3 for reference). Figures 2and 3 summarize these data, and for simplicity, the enzymeswere ranked according to their susceptibilities to clavulanicacid.

Figure 2 compares the IC50s of clavulanic acid with those oftazobactam and sulbactam for the conventional-spectrum plas-mid-mediated P-lactamases. Figure 2a shows that clavulanicacid was more potent than sulbactam against almost all of theenzymes in this group. OXA-2 was the only enzyme againstwhich sulbactam demonstrated better activity than clavulanicacid. Statistical analysis of the IC50s demonstrates that, overall,clavulanic acid was 20 times more potent than sulbactamagainst these 15 enzymes (Table 4).

Figure 2b shows that tazobactam and clavulanic acid hadsimilar activities against the 15 conventional-spectrum en-zymes. Analysis of the IC50s failed to reveal a significantdifference in overall potency between the two inhibitors andthis group of enzymes (Table 4). The comparison of panels aand b of Fig. 2 clearly demonstrates that tazobactam was morepotent than sulbactam. In fact, overall, tazobactam was signif-icantly more potent than sulbactam against this group ofenzymes (Table 4).

Clavulanic acid was also a more potent inhibitor thansulbactam for 19 of the 20 extended-spectrum plasmid-medi-ated ,B-lactamases (Fig. 3a). TEM-3 was the only extended-spectrum enzyme against which the activity of sulbactam wascomparable to that of clavulanic acid. Statistical analysis of theIC50s shows that, for the 20 enzymes studied, clavulanic acidwas 14 times more potent overall than sulbactam (Table 4).

Figure 3b compares the ,B-lactamase inhibitory activities of

clavulanic acid and tazobactam against 20 TEM- and SHV-derived extended-spectrum P-lactamases. Both inhibitors werehighly active, with IC50s of less than 0.5 FM. In terms of overallpotency, the difference between them was not statisticallysignificant.

DISCUSSIONDetailed kinetic studies have been essential in elucidating

the mode of action of mechanism-based P-lactamase inhibitors(5, 21). However, because of the complexities of such interac-tions, these studies have been confined to a limited numker ofrepresentative 1-lactamases. The proportion of clinical isolatesproducing plasmid-mediated 1-lactamases is high (37), and thediversity of these enzymes is also expanding as an inevitableconsequence of microbial adaptation (27). Consequently, froma clinical perspective, it is also essential to investigate theactivities of the commercially available ,-lactamase inhibitorsagainst a broad variety of clinically derived ,B-lactamases.Therefore, we have determined the IC50s of the three com-mercially available 13-lactamase inhibitors for 35 plasmid-mediated 3-lactamases. IC50s measure only the activity of a,-lactamase inhibitor at a fixed time interval after incubationwith an enzyme (5 min in this study). The values obtained aredependent on various kinetic parameters associated with theinteractions of both inhibitor and substrate with P-lactamase.Nevertheless, IC50s do provide the most practical way ofevaluating the relative activities of P-lactamase inhibitors forsuch a large number of enzymes. For these reasons, manyother groups have adopted IC50 assays as a means of charac-terizing 13-lactamases (5, 19, 31).

770 PAYNE ET AL.

COMPARATIVE ACTIVITIES OF INHIBITORS OF ,B-LACTAMASES

10 100

*---------o Erzyme-B

*----------- DJP-1*--------------- SHV-5

*--------- TEM-E3TEM-3

*---------o TEM-10*-------------0 TEM-5

*----------0 Enzyme-D*--------------- 0 SHV-3

*-0-------- TEM-El

*----------------4 SHV-2--------- TEM-E4*----o Enzyme-A

*-----------) TEM-E2

-------------- Enzyme-E

-------0 TEM-7

*---- -o TEM-6

'- 0 CAZ-3*- - - o TEM-9*------------o Enzyme-C

b I50 (JIM)0.01 0.1 1 10 100

o Ernyme-B*- DJP-1------0 SHV-5*-- TEM-E30---o TEM-100-------o TEM-5*-- -o SHV-3*--- SHV-2

oc Enzyme-E& -o TEM-7

@0 TEM-6@0 TEM-9

TEM-3 o ---oErmymeD - - - --

TEM-EI 0- --0TEM-E4 0-@TEM-E2 - 4Ernyme-A 04CAZ-3 0--0

Enzyme-C ----*

FIG. 3. Comparison of the IC50s of 3-lactamase inhibitors against TEM- and SHV-derived extended-spectrum ,B-lactamases. (a) IC5os ofclavulanic acid (0) and sulbactam (0); (b) IC50s of clavulanic acid (0) and tazobactam (0).

The semiautomated method for the determination of IC50sproved to be a rapid method for comparing the activities of,-lactamase inhibitors with an acceptable level of reproduc-ibility.These results show that clavulanic acid was a more potent

inhibitor than sulbactam of both conventional-spectrum andextended-spectrum TEM and SHV enzymes. Jacobs et al. (15)determined the MICs of ampicillin in combination with clavu-lanic acid and sulbactam for 12 strains of E. coli producingconventional-spectrunm 3-lactamases, and although IC50s andMICs cannot be directly correlated, the MICs do suggest thatclavulanic acid had greater activity than did sulbactam againstconventional-spectrum enzymes. The only enzyme for whichthe IC 0 of sulbactam was lower than that of clavulanic acidwas OXA-2. However, OXA-2 is of minor clinical significance,being produced by

AN11MICROB. AGENTS CHEMOTHER.

mase inhibitors has also been reported with MIC determina-tions conducted on 184 ampicillin-resistant clinical isolates (2).This demonstrates that clavulanic acid and tazobactam arepotent inhibitors not only of the conventional-spectrum ,B-lac-tamases but also of the newer enzymes.

ACKNOWLEDGMENTS

We thank Brian Bond for statistical evaluation of the data and ChrisBlackmore for photographic work.

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