ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1991, 0066-4804/91/091900-05$02.00/0 Copyright ©D 1991, American Society for Microbiology

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Vol. 35, No. 9

Mechanism of Action of BAY v 3522, a New Cephalosporin with Unusually Good Activity against Enterococci GUIDO AMALFITANO,' A. GROSSATO,2 AND R. FONTANA'* Institute of Microbiology, University of Verona, 37134 Verona,' and Institute of Microbiology, University of Padua, 35100 Padua,2 Italy Received 19 March 1991/Accepted 25 June 1991

The in vitro activity of BAY v 3522, a new cephalosporin with unusually good activity against enterococci, was tested on 100 clinical isolates of Enterococcusfaecalis. The MIC for 86.3% of the strains was 4 ,ug/ml, whereas the MIC for 13.7% ranged from 8 to 16 ,ug/ml. No differences were found between MICs determined with low- or high-density inocula. The bactericidal activity of BAY v 3522 was tested on eight clinical strains; most strains showed a ca. 3-log decrease of the original inoculum at two to eight times the MIC. The interaction of BAY v 3522 and of other beta-lactams with penicillin-binding proteins (PBPs) was studied with a laboratory strain, E. hirae ATCC 9790, producing a discernible amount of PBP 5, a protein belonging to the family of low-affinity PBPs, responsible for the low susceptibility of enterococci to beta-lactams. PBPs 3 and 5 of ATCC 9790 showed the highest affinity for the new cephalosporin. Bay V 3522 at the MIC (8 ,ug/ml) saturated these two PBPs without any significant binding to the other PBPs. This result may explain the good antienterococcal activity of BAY v 3522.

Cephalosporins exert only weak activity against enterococci (10, 17-19, 21-23). This resistance has been associated with the very low affinity of these beta-lactams for enterococcal penicillin-binding proteins (PBPs) and, in particular, for a high-molecular-weight PBP which has been suggested as taking over the functions of the other PBPs when these are inactivated by mutations or beta-lactams (3-5, 8, 11, 12, 15, 20, 24, 26). The role of this PBP in cell physiology and in the mechanism of resistance to beta-lactams in enterococci has been extensively studied in Enterococcus hirae (formerly E. faecium) ATCC 9790, in which this protein corresponds to PBP 5 (5, 12). However, PBPs with properties similar to E. hirae PBP 5 also have been shown to be present in strains of E. faecalis and E. faecium, the most important enterococcal species for human pathology (12, 24, 26). Recent publications (7, 16, 25) suggest that a new oral cephalosporin, BAY v 3522 {7-[D-2-amino-2-(2-aminobenzothiazol-6-yl)-acetamido]-3-[(Z)-1-propen-1-yl]-3-cephem-4carboxylic acid monohydrate}, exhibited unusually good activity against gram-positive cocci, including enterococci. Therefore, we focused our study on (i) the in vitro activity of this new cephalosporin against clinical isolates of enterococci compared with the activities of other agents and (ii) the interaction of this antibiotic with PBPs of E. hirae ATCC 9790. We found that BAY v 3522 exhibited an unusually higher affinity for PBP 5 than for other PBPs, in contrast to the behavior exhibited by other beta-lactams (5). MATERIALS AND METHODS Bacterial strains. One hundred enterococcal clinical isolates obtained from different body areas were used in this study. These isolates were initially identified as enterococci by growth in 6.5% salt broth and by hydrolysis of esculin in the presence of 40% bile (6). Subsequent identification was performed with the API 205 system (Analytab Products, Plainview, N.Y.). E. faecalis 1310, kindly provided by R. C. Moellering, Jr., was a clinical isolate recovered at the *

Corresponding author.

Massachussetts General Hospital, Boston. E. hirae ATCC 9790 was a collection strain which is susceptible to penicillin. E. hirae R40, a mutant which is highly resistant to penicillin and which overproduces PBP 5, was isolated from ATCC 9790. E. hirae Revl4, which does not synthesize PBP 5, was a penicillin-hypersusceptible derivative of R40 (12, 14). The experiments were carried out by growing bacteria in Mueller-Hinton broth (Difco Laboratories) or Mueller-Hinton broth containing 1.5% agar (Mueller-Hinton agar). Antibiotics and reagents. Antibiotics were obtained from the following sources: BAY v 3522, Bayer A.G.; cefixime, Lederle Laboratories (Fareham, England); cefuroxime, Glaxo (Greenford, England); cefaclor, Eli Lilly (Indianapolis, Ind.); and ampicillin, E. R. Squibb & Sons. 3H-benzylpenicillin ethylpiperidinium salt (specific activity, 25 Ci/ mmol) was kindly supplied by Merck & Co., Inc. (Rahway, N.J.). All other chemicals were commercially available reagent-grade products. In vitro susceptibility testing. MICs were determined by an agar dilution technique with Mueller-Hinton agar. Overnight cultures, diluted 1:100 or undiluted, were transferred to the surface of the antibiotic-containing agar with a multipoint inoculating device to yield a final inoculum of 104 or 106 CFU per spot, respectively. Plates were read after 24 h of incubation at 37°C. The MIC was defined as the lowest concentration inhibiting the formation of colonies. The MBC was determined as described by Fontana et al. (13) with a macrodilution technique consisting of the preparation of glass tubes containing serial twofold dilutions of antibiotic in 5 ml of Mueller-Hinton broth. The tubes were inoculated with overnight cultures to obtain a final density of ca. 5 x 105 CFU/ml. After 20 h of incubation at 37°C, all tubes were vortexed; 4 h later, all tubes were examined for visual turbidity and the MICs were recorded. After the tubes were vortexed again, a 0.1-ml sample was taken from each tube without visual turbidity and serially diluted, and 0.1 ml of each dilution was spread onto Mueller-Hinton agar plates. CFU were counted after 24 h of incubation. For killing curves, the same procedure as that used for MBC determi1900

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MECHANISM OF ACTION OF BAY V 3522

TABLE 1. Comparative in vitro activities of BAY v 3522 and other antibiotics % of strains inhibited at the following concn (,ug/ml):

Antibiotic 1

BAY v 3522 Ampicillin Cefaclor Cefuroxime Cefixime

15

2

4

8

16

2 6

13

64

85 15

32

64

128

BAY v 3522

E. faecalis strain

nations was used, but CFU counting was done at 0, 2, 4, 6, and 24 h of incubation. PBP assays. The binding of beta-lactams to PBPs was investigated with E. hirae ATCC 9790. Samples (20 ml) of a culture in the early exponential phase of growth (4 x 107 bacteria per ml) were incubated with various antibiotic concentrations for 60 min at 37°C (12). Under these conditions, growth was inhibited to various extents, depending on the antibiotic concentrations, but culture lysis did not occur. A sample containing approximately 4 mg of bacteria (dry weight) was taken after incubation with the antibiotic, centrifuged, and suspended in 0.1 ml of 10 mM phosphate buffer (pH 7). The cell suspension was incubated with 100 KM 3H-benzylpenicillin for a time equal to that used for binding with cold beta-lactam. Membrane isolation and sample preparation for gel electrophoresis were done as described previously (12). The amount of radioactivity bound to PBPs was estimated by microdensitometry of the autoradiograms with a Shimadzu DR-2 densitometer. RESULTS In vitro susceptibility studies. The activity of BAY v 3522 against 100 clinical isolates of E. faecalis is shown in Table 1. The MIC range for BAY v 3522 was 4 to 16 ,ug/ml, and the MICs for 50% of isolates (MlC50) and for 90% of the isolates (MIC90) were 4 and 16 ,ug/ml, respectively. No difference was found between MICs determined with low- and highdensity inocula. All isolates proved to be highly resistant to the other cephalosporins, the MIC90 being >128 ,ug/ml. As expected, ampicillin showed the best activity against all isolates (MIC50, 2 pKg/ml; MIC90, 4 ,ug/ml; MIC range, 1 to 8

,ug/ml).

The bactericidal activities of BAY

TABLE 2. Loglo reduction in CFU after 24 h of incubation in presence of various concentrations of BAY v 3522 and ampicillin above the MIC

>128

100 100 100

v

3522 and ampicillin

were tested against eight clinical isolates. Table 2 shows that seven of eight clinical isolates exhibited a paradoxical re-

sponse to the bactericidal activities of BAY v 3522 and ampicillin, being more susceptible to the killing activity of low than of high antibiotic concentrations. At two times the MIC, the percentages of survival ranged from 0.1 to 0.9% with BAY v 3522 and from 0.1 to 0.4% with ampicillin. At two to eight times the MIC, most isolates showed a ca. 3-log decrease of the original inoculum. Isolate 3678 did not exhibit a paradoxical response and was efficiently killed by both antibiotics at all concentrations above the MIC. This isolate was found to produce a large amount of an autolytic enzyme, which was responsible for the highest susceptibility to high antibiotic concentrations (9). The bactericidal activity of BAY v 3522 was also tested by killing-curve assays for four of the eight clinical isolates. The results obtained paralleled those obtained by the MBC test. After 24 h of incubation, isolates E6, 3678, and 1310 were approximately 99.9% killed by two times the MIC, whereas the CFU of

1901

E6 3678 263.61 263.1 258.3 270.52 24 1310

Ampicillin

Log1o reduction of

Log1o reduction of MIC (p.g/ml)

4 8 4 8 4 4 4 4

CFU at MIC multiple of': 2 8 32

2.96 2.70 3.15 2.32 2.05 3.52 2.70 2.35

2.77 2.09 3.30 2.66 2.70 1.26 1.77 1.21 1.08 0.26 2.62 1.29 2.40 1.52 2.70 1.62

MIC (,ug/ml)

CFU at MIC multiple of a:

2 1 1 1 2 1 1 1

3.40 2.80 1.92 2.52 2.52 2.52 2.40 2.00 1.49 2.70 1.60 1.51 3.05 1.51 1.35 2.40 2.52 1.07 2.40 2.52 1.07 2.70 2.52 1.07

2

8

32

" The log1o reduction of the initial cell population at the various antibiotic concentrations was determined after 24 h of incubation at 37'C as described in Materials and Methods.

isolate 258.3 decreased by 99.2%. At increasing antibiotic concentrations, all isolates, except 3678, exhibited a paradoxical response. Within a shorter incubation time (6 h), only the killing kinetics of isolate 1310 approached the maximum value (data not shown). Interaction of beta-lactams with PBPs and effects on cell growth. To determine the interaction of beta-lactams with PBPs, we used E. hirae ATCC 9790, a laboratory strain extensively used in studies on the mechanism of action of penicillin against enterococci (3-5, 8, 10-12, 14, 20). This strain produces a relatively low amount of PBP 5 and shows a susceptibility to beta-lactams similar to that of E. faecalis clinical isolates (Table 3). In comparison with the activity of the other beta-lactams, the activity of BAY v 3522 was decreased against strain R40, which was also resistant to ampicillin, and increased against strain Revl4, which was also susceptible to the other beta-lactams. To correlate the saturation of PBPs with the inhibitory and bactericidal activities of BAY v 3522 and the other betalactams, we performed competition experiments with growing cells of strain ATCC 9790 treated with one-half, one, and two times the MIC for 60 min at 37°C and thereafter with a saturating concentration of radioactive penicillin. As shown in Fig. 1A, BAY v 3522 at the MIC (8 ±Lg/ml) bound 95% of PBP 3 and 81% of PBP 5, inhibited the binding of radioactive penicillin to PBPs 2 and 4 by 61 and 74%, respectively, and did not significantly interact with PBPs 1 and 6, as the percentages of binding were 13 and 29%, respectively. Ampicillin (Fig. 1B) showed a higher affinity than did BAY v 3522 for all PBPs and, at the MIC (2 ,ug/ml), bound 96% of PBP 2 and 97% of PBPs 3, 4, and 5. At one-half times the MIC this antibiotic bound the same amounts of PBPs 2, 3, 4, and 6 as it did at the MIC but only bound 12% of PBP 5. The MIC of cefaclor (64 p.g/ml) saturated 98% of PBP 5 and bound 83% of PBP 1 and 58% of PBP 6, whereas PBPs 2, 3, and 4 were all saturated 95% by one-fourth times the MIC (Fig. 2A). Cefuroxime showed a low affinity for all PBPs, inhibited growth at a very high concentration (MIC, 512 p.g/ml) and, at this concentration, apparently bound 52% of PBP 2, 60% of the PBP 3-PBP 4 complex, and 61% of PBP 5. PBPs 1 and 6 were practically unaffected even by two times the MIC (Fig. 2B). Killing curves for ATCC 9790 treated with the four betalactams were also determined under the same conditions as

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AMALFITANO ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 3. Beta-lactam susceptibilities of E. hirae ATCC 9790, R40, and Rev14 MIC (1Lg/ml) of:

E. hirae strain

synthesisa

PBP 5

BAY v 3522

Ampicillin

Cefaclor

Cefuroxime

ATCC 9790 R40 Revl4

+ +++ -

8 64 0.5

2 32 0.015

64 256 0.5

512 1,024 0.5

Cefixime

.2,048 .2,048 0.5

aSee reference 14.

those used in the PBP studies. Ampicillin showed the best bactericidal activity; at two times the MIC, a concentration at which most PBPs were saturated, this drug caused a 2-log decrease in the initial population within 6 h and more than a 3-log decrease after 24 h of incubation (Fig. 3A). At two times the MIC, BAY v 3522 was less bactericidal, causing 95.9 and 99.8% decreases in the initial population after 6 and 24 h, respectively (Fig. 3B). As shown in Fig. 1A, BAY v 3522 at this concentration saturated only PBPs 3 and 5. However, at increasing concentrations of antibiotic, which presumably saturated more PBPs, the killing kinetics did not increase significantly. Because of the low antibacterial activities, the bactericidal activities of cefaclor and cefuroxime were evaluated at very high concentrations. The killing kinetics of cefaclor did not significantly differ when two and eight times the MIC (128 and 512 pug/ml, respectively) were used (Fig. 3C). Cefuroxime exhibited better bactericidal activity at eight times the MIC (4,096 p,g/ml) than at two times the MIC (1,024 ,ug/ml), a concentration which inhibited growth but did not saturate PBP 5 or any other PBP (Fig. 3D). DISCUSSION

The in vitro susceptibility of clinical isolates of E. faecalis to BAY v 3522 determined in this study confirmed the unusually good activity of this new oral cephalosporin against a bacterial species well known for its natural poor susceptibility to these beta-lactams. The MICs of the antibiotic were two- to four-fold higher than the MICs of ampicil-

lin, the beta-lactam with the highest activity against enterococci, but to the best of our knowledge were far lower than those of all cephalosporins included in this study and previous studies (2, 15, 18, 22, 23). The relatively good activity of BAY v 3522 in inhibiting the growth of E. faecalis was also accompanied by relatively good bactericidal activity, as most strains showed a ca. 3-log decrease of the original inoculum at two to eight times the MIC. Like ampicillin, the new cephalosporin killed most E. faecalis strains in a paradoxical way. However, it has been shown recently that this phenomenon depends on the conditions used for MBC determinations, and it has been suggested that the paradoxical response cannot influence the in vivo activity of a beta-lactam which may have shown good in vitro bactericidal activity at concentrations which are easily reached in the infected body area (13). Previous studies have shown that the good antienterococcal activity of certain beta-lactams is due to their ability to combine at relatively low concentrations with most or all of the PBPs of these bacteria (5, 8, 10, 14, 15, 24, 26), including a particular PBP characterized by a very low affinity for these antibiotics. These proteins are produced in different amounts by enterococcal strains and are thought to be responsible for the low susceptibility and high resistance of enterococci to these antibiotics. It has been postulated that under certain growth conditions, the low-affinity PBPs can take over the functions of the other PBPs, thus becoming the most important targets for both growth inhibition and killing (12, 20). The study of the interaction of BAY v 3522 with PBPs of

vI-a

7 7~ .7.77:. ". _B7. ~~~~~~~~~~~~

FIG. 1. Interaction of BAY v 3522 (A) and ampicillin (B) with PBPs on E. hirae ATCC 9790 cells growing in the presence of various antibiotic concentrations. The MICs of BAY v 3522 and ampicillin were 8 and 2 ,ug/ml, respectively. Numbers across the bottom indicate antibiotic concentrations. C, untreated culture.

FIG. 2. Interaction of cefaclor (A) and cefuroxime (B) with PBPs growing in the presence of various antibiotic concentrations. The MICs of cephaclor and cefuroxime were 64 and 512 Lglgml, respectively. Numbers across the bottom indicate antibiotic concentrations. C, untreated culture. on E. hirae ATCC 9790 cells

VOL. 35, 1991

MECHANISM OF ACTION OF BAY V 3522

A

1000000

B

1000000

100000

1903

100000

CFU/mi

CFU/mi

10000

10000

1000

1000

100 Oh

100 2 h

4 ]h

6 h

0 h

24 h

C

4 h

6 h

24 h

D

1000000 I

1000000 I

100000

100000

CFU/ml

CFU/al

10000

10000

1000

1000

100 Oh

2 h

time (hours)

time (hiours)

2 h 4 h 6 h time (hours)

24 h

100 1Oh

2 h 4 h 6 h time (hours)

24 h

FIG. 3. Bactericidal activities of ampicillin (A), BAY v 3522 (B), cefaclor (C), and cefuroxime (D) for E. hirae ATCC 9790 at 2 (a), 8 (O), and 32 (*) times the MIC.

growing cells of E. hirae ATCC 9790 showed that at the MIC, this antibiotic saturated PBPs 3 and 5 but none of the other PBPs (Fig. 1A). Under the conditions used, PBP 5 behaved as the most important target for growth inhibition by the antibiotic, as the saturation of PBP 3 alone (the only PBP saturated at one-half times the MIC) was not sufficient to inhibit growth. These results reveal a particular property of the new cephalosporin never exhibited by any of the beta-lactams developed up to date, i.e., an affinity for PBP 5 which is higher than that for the other PBPs (with the exception of PBP 3). PBP 5 of E. hirae and, more recently, low-affinity PBPs of E. faecium have been suggested as also being important targets for the bactericidal activity of beta-lactams (1, 20). Complete saturation of PBP 5 was associated with cell death in E. hirae ATCC 9790, although it was not possible to establish whether it was saturation of all PBPs or only of PBP 5 that was needed for cellular killing, because, as PBP

5 showed the lowest affinity for beta-lactams, its saturation was observed only after all the other PBPs had been saturated (20). The finding that the rate of killing caused by BAY v 3522 at two times the MIC, which saturated PBP 5 but not the other PBPs (except for PBP 3), was similar to the rate of killing caused by BAY v 3522 at concentrations above two times the MIC, which presumably saturated most of the other PBPs (Fig. 3B), suggested that saturation of PBP 5 was the event required for triggering cellular killing. This conclusion was further supported by the finding that all betalactams exhibited maximum bactericidal activities at concentrations at which PBP 5 was saturated (whether the other PBPs were saturated or not); these concentrations were two times the MIC for ampicillin and cefaclor and more than eight times the MIC for cefuroxime. The improved activity of BAY v 3522 against grampositive cocci has been associated with the substituent on

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ANTIMICROB. AGENTS CHEMOTHER.

AMALFITANO ET AL.

the beta-acyl side chain, an aminobenzothiazol grouping (25) also responsible for its good stability against many P-lactamases. This substituent may confer a specific increased affinity for PBP 5 on the cephem nucleus, this being a property which to date has never been shown by any other cephalosporin. This drug may therefore represent a step forward in the design of new, more effective beta-lactams. ACKNOWLEDGMENTS This study was supported in part by grant 90.00038.70 from Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie e Biostrumentazione, and in part by a grant from Bayer AG. We are indebted to P. J. Cassidy of Merck, Sharp and Dohme for the generous gift of 3H-benzylpenicillin.

13.

14.

15.

16. 1.

2. 3.

4.

5.

6. 7.

8. 9.

10.

11.

12.

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