Journal of Antimicrobial Chemotherapy (1990) 25, 567-574

Inhibition of /Mactamases by tazobactam and in-vitro antibacterial activity of tazobactam combined with piperadllin Fnsahiro HlgashiUni,** Aldo Hyodo,* Naofnmi Ishida,6 Matsuhisa Inoue*

and Susumu Mrtsahashi"

The in-vitro synergistic activity of tazobactam, a new /Mactamase inhibitor, combined with piperacillin was tested against various /Mactamase-producing strains. The /Mactamase inhibitory activity of tazobactam against various known types of filactamase was also tested in comparison with clavulanic acid or sulbactam. Tazobactam caused a remarkable reduction of the piperacillin MICs for peniciUinaseand oxyiminocephalosporinase-producing strains and also showed a moderate synergistic effect against cephalosporinase-producing strains. The bactericidal activity of pipcracillin was enhanced in combination with tazobactam. Tazobactam inhibited the penicillinases, and the oxyiminocephalosporinase produced by Proteus vulgaris, at low concentration. In these cases its activity was comparable with that of clavulanic acid and stronger than that of sulbactam. Tazobactam demonstrated a better' inhibitory capability than sulbactam against the cephalosporinases tested. Tazobactam was able to inactivate intracellular /Mactamase in Prot. vulgaris and Morganella morganii, confirming its ability to penetrate the cell membrane of these species. Introduction

Bacterial strains resistant to /Mactam antibiotics have been increasing in frequency. The main factor in this resistance is thought to be the production of /Mactamases. Many new semisynthetic /Mactam agents resistant to /Mactamase have been developed in attempts to overcome this problem. Also, in recent years, attention has been paid to compounds with very little intrinsic activity, but with the ability to irreversibly inhibit /Mactamases. In the treatment of infections caused by /Mactamase-producing strains, /Mactamase inhibitors combined with /Mactams have been useful. Such inhibitors are clavulanic acid, halopenicillanic acids, and penicillanic acid sulphones, including sulbactam and tazobactam (YTR-830H) (Brown et al., 1976; Reading & Cole, 1977; English et al., 1978; Paisley & Washington, 1978; Matsuura et al, 1980; Gutmann et al., 1986). Clavulanic acid combined with amoxycillin or ticarcillin, and sulbactam with ampicillin have been approved for clinical use. The purpose of this present study was the evaluation of tazobactam. Its inhibitory activity against various /Mactamase was compared with those of clavulanic acid or sulbactam, and an evaluation of the synergistic activity of tazobactam combined with piperacillin was undertaken against various /Mactamase-producing strains. •Corresponding author c/o Episome Institute, 2220 Fujimi-mura kogure, Seta-gun, Gunma, Japan. 0305-7453/90/040567 + 08 $02.00/0

567 ©1990 The British Society for Antimicrobial Chemotherapy

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'Episome Institute, Fujimi-mura, Seta-gun, Gunma, bTaiho Pharmaceutical Co. Ltd, Tokushima and 'School of Medicine, Gunma University, Maebashi, Gunma, Japan

568

F. HigasfaHanl et aL Materials and methods

Compounds

Piperacillin was provided by Toyama Chemical Co., Toyama, Japan. The /Mactamase inhibitors were tazobactam (Taiho Pharmaceutical Co., Tokushima, Japan), clavulanic acid (Beecham Yakuhin Co., Tokyo, Japan) and sulbactam (Pfizer Taito Co., Tokyo, Japan). The following antibiotics were commercially available products: penicillin G, cephaloridine and cephalothin. Organisms

Determination of MICs MICs were determined by the agar dilution method with doubling dilutions of antibiotics. The medium used for MIC determination was Sensitivity Disk Agar-N (Nissui Seiyaku Co.). The test organisms were first grown overnight at 37°C in Sensitivity Test Broth (Nissui Seiyaku Co.). These cultures were diluted with buffered saline gelatin, and an inoculum of about 103 cfu per spot was applied to the drugcontaining agar plates. The MICs were recorded after 18 h of incubation at 37°C as the lowest concentrations of drug that inhibited visible growth of bacteria. Bactericidal activity An overnight culture of the test organism in Antibiotic Medium 3 (Difco) was diluted to about 104 cfu/ml with fresh broth, and the diluted culture was incubated for 2 h with shaking at 37°C. Piperacillin or the combination of piperacillin with tazobactam was added to parallel cultures, which were incubated again with shaking at 37°C. Samples were obtained at 2, 4, 6 and 8 h after addition of the drugs. The samples were serially diluted in saline and the number of viable cells was determined by counting the number of colonies formed when 01 ml of an appropriate dilution was mixed with drug-free Sensivity Disk Agar-N, poured into plates and incubated for 18 h at 37°C. Preparation of fi-lactamase

Bacteria were grown overnight at 37CC in Brain Heart Infusion Broth (Nissui). The cultures were diluted ten-fold with fresh Medium B (Difco) and grown at the same temperature. Cultures of penicillinase-producing bacteria were harvested by centrifugation after incubation for 5h. Cultures of cephalosporinase or oxyiminocephalosporinase-producing bacteria were incubated for 3 h, after which cefoxitin was added as an inducer. After a further 2 h of incubation, the cells were harvested by centrifugation. The harvested organisms from either method were washed twice with 50 nut phosphate buffer (pH 7), then disrupted by sonic treatment in an ice bath. The crude extracts were centrifuged for 30min at 12,000 rpm at 4°C, and streptomycin (2% w/v) was added to the supernatant which was then dialysed overnight against distilled water. This extract was purified by column chromatography or diethylaminoethyl-cellulose or carboxymethyl-Sephadex.

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The organisms used were reference strains stored in our laboratories, and recent clinical isolates collected from various hospitals in Japan.

Tazotmctam and ptperacflttn

569

fl-Lactamase assays

^-Lactamase activity was determined by spectrophotometry at 30cC, in 005 M phosphate buflFer. The inhibitory activity of /Mactamase inhibitors was measured by the same method. For hydrolysis by /Mactamase, penicillin G, cephaloridine and cephalothin were used as substrates. The percent inhibition was calculated as follows; percent inhibition = (A-B)/A x 100, where A was the hydrolysis rate in the uninhibited reaction and B was the hydrolysis rate in the inhibited reaction. The ID*, (inhibitor concentration inhibiting 50% of the enzyme activity) was calculated from a plot of percent inhibition versus inhibitor concentration.

Bacteria were grown overnight at 37°C in Brain Heart Infusion Broth. The culture was diluted ten-fold with the same broth and grown at the same temperature for about 3 h. The cells were harvested by centrifugation and resuspended in saline at 37°C and the inhibitor added. At intervals of 1, 2, 4 and 6 h after addition of the inhibitors, samples were withdrawn and the bacteria were harvested. All the harvested organisms were washed twice with 50 mM phosphate buffer and suspended in 2 ml of phosphate buffer. The cells were then disrupted by sonic treatment in an ice bath. Debris was removed by centrifugation for 10 min at 12,000 rpm and 4°C, and the supernatant was used as a

Table L Combined antibacterial activity of piperacillin with tazobactam, clavulanic acid and sulbactam against penicillinase-producing strains

Strains

piperacillin alone

Each coli ML4901" ML4901/Rms212(TEM-l) ML4901/Rms213(OXA-l) ML4901/Rtcl6 (OXA-2) ML4901/Rmsl49(PSE-l) ML4901/TEM-1 ML4901/TEM-2 ML4901/OXA-1 ML4901/OXA-2 ML4901/PSE-1 ML4901/PSE-3 ML4901/SHV-1

>200 100 50 >200 >200 >200 200 >200 >200 200 >200

Staphylococcus aureus ML15009* ML15009/pI258 K.

MIC (mg/1) piperacillin with tazobactam clavulanic acid

313 313 12-5 313 1-56 313 313 12-5 1-56 313 1-56 12-5

1-56 1-56 6-25 1-56 1-56 1-56 1-56 6-25 1-56

0-1 >200 >200

313

sulbactam

1-56 12-5

25 313 1-56 1-56 >200

25 1-56

313

200

1-56 6-25

1-56 >2O0

0-39

0-20

1-56

313

1-56

pneumoniae

GN69

"Pcnidllinaje non-producing host strain. All inhibitors were present at a concentration of 5 mg/L

50

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Inhibition of intracellular fi-lactamase

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Table II. Combined antibacterial activity of piperacillin with tazobactam, davulanic acid and sulbactam on cephalosporinase- and oxyirninocephalosporinasc-producing strains

Strains

Inducibility*

piperacillin alone

Esch. coli GN5482 Esch. coli GN14930 Ent. cloacae GN7471 Ent. cloacae GN5797 C.freundii GN346 C.freundii GN7391 Providencia rettgeri GN4430

C

25

C I I I I I

50 50 25 100 >200 6-25

PTOV. rettgeri GN4762

I

12-5

Ser. marcescens GN10857 Ser. marcescens GN 14932 M. morganU GN5407 M. morganii GN5375 Ps. aeruginosa GN 10362 Ps. aeruginosa GN10367 Prot. vulgaris GN7919* Ps. cepacia GNU 164'

I I

>200 12-5

I

6-25

I I I I I

6-25 25 >200 >200 25

1 , Inducible; C, constitutive. *Oxyiminocephalosporinase producing strain. AD inhibitors were present at a concentration of 3 mg/L

MIC (mg/1) piperacillin with tazobactam clavulanic acid 1-56 6-25 12-5 12-5 12-5 >200 1-56

313 200 6-25 0-39 0-39 6-25

25

25 25 100 50 >200

313 6-25

200 50 25

50 50

25 313

200 1-56

0-20

313

sulbactam 1-56

313 6-25 12-5

100 >200 313 313 200 6-25 0-78 0-78 6-25

100

313 0-20

Tazobactam and piperadDfai

571

source of /Mactamase. The remaining enzyme activities was measured by the spectrophotometric method.

Results Combined antibacterial activity of piperacillin with f}-lactamase inhibitors

Bactericidal activity The in-vitro bactericidal activity of piperacillin with tazobactam was determined by counting the number of viable cells after incubation with piperacillin alone and in combination with tazobactam (Figure I). Against the TEM-1 /Mactamase-producing Escherichia coli strain ML5005, strong bactericidal activity of piperacillin at 313 or 6-25 mg/1 combined with 5 mg/1 of tazobactam was observed. By contrast, piperacillin alone (313, 6-25 mg/1) could not inhibit the growth of the bacteria. Against Klebsiella pneumoniae ML5O06, which produced a Class IV /Mactamase, the bactericidal activity of piperacillin (12-5, 25 mg/1) with tazobactam (5 mg/1) was stronger than 50 mg/1 of piperacillin alone. For M. morganii GN14606, piperacillin at 1-56 or 3-13 mg/1 could inhibit the growth of bacteria when combined with tazobactam (5 mg/1), but not alone at 12-5 mg/1.

Inhibition of fl-lactamase activity The 50% inhibitory doses (IDJO,) of tazobactam, clavulanic acid and sulbactam against various /Mactamases are presented in Table HI. The penicillinase Type I ( = TEM-1) (Mitsuhashi & Inoue, 1981) was inhibited more strongly by tazobactam than by clavulanic acid and sulbactam. The inhibitory activity of tazobactam was comparable with that of clavulanic acid and stronger than that of sulbactam for staphylococcal penicillinase (Type V: Mitsuhashi & Inoue, 1981), the chromosomal penicillinase from

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The antibacterial activity of piperacillin with 5 mg/1 of tazobactam against bacteria producing known types of /Mactamase was compared with that of piperacillin with 5 mg/1 of clavulanic acid or sulbactam and with piperacillin alone (Table I). All penicillinase-producing strains tested were resistant to piperacillin (MIC, 50- > 200 mg/1). However in combination with tazobactam piperacillin was effective at < 12-5 mg/1 against these strains. This synergistic effects of tazobactam with piperacillin was comparable with that of clavulanic acid and better than that of sulbactam. Table II shows the combined effect of piperacillin tazobactam against cephalosporinase- and oxyiminocephalosporinase-producing strains. Tazobactam showed synergistic effects for some cephalosporinase-producing strains. With tazobactam (5 mg/1) the MICs of piperacillin for these strains were reduced to < 12-5 mg/1, except with Citrobacter freundii GN7391, Serratia marcescens GN10857 and Pseudomonas aeruginosa GN10367. Sulbactam behaved similarly to tazobactam on the other hand, the potentiating effects of clavulanic acid were minimal against these strains, and clavulanic acid antagonized piperacillin effects against Enterobacter cloacae GN5797 and Morganella morganii. All three inhibitors significantly reduced the MIC of piperacillin for Proteus vulgaris and Ps. cepacia strains.

572

F. Higashhmni et aL

o

4 Tim* (h )

6

6

Figure 1. Bactericidal activity of piperacillin with tazobactam on 0-lactamase producing strains, (a) Esch. coli ML5005; (b) K. pneumoniae ML5006; (c) M. morganii GN14606. O, Control without drugs; D , with piperacillin alone at 3-13 mg/1 (a), 12-5 mg/1 (b), 1-56 mg/1 (c); O, with piperacillin alone at 6-25 mg/1 (a), 25 mg/1 (b), 313 mg/1 (c); A , with piperacillin alone at 25 mg/1 (a), 50 mg/1 (b), 12-5 mg/1 (c); • , combined piperacillin i t 3-13 mg/1 («), 12-5 mg/1 (b), 1-56 mg/1 (c); A , combined piperacillin at 6-25 mg/1 (a), 25 mg/1 (b), 3-13 mg/ml (c) with tazobactam. The inhibitor concentration was 5 mg/1 in all cases.

K. pneumoniae GN69 and oxyiminocephalosporinase from Prot. vulgaris GN7919. Furthermore, tazobactam also showed better inhibitory activities than sulbactam against the different cephalosporinases tested. Inhibition of intraceUular fi-lactamase

The inhibition of intraceUular ^-lactamase by tazobactam was examined using Prot. vulgaris and M. morganii (Figure 2). The bacterial cells were incubated in saline with each /Mactamase inhibitor, and the remaining enzyme activity was measured at intervals. The enzyme in whole cells of Prot. vulgaris GN919 or M. morganii GN5407

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-z

Tazobactam and piperacflltn

573

(o)

4

6

1 2

4

6

I

2

Incubation tlmt ( h )

Ftgare 2. Inhibition of intracelhilar /J-lactamaie. (a) Proi. vulgaris GN7919 (4-4 x 101 oeOj/ml); (b) M. morganti GN5407 (6-3 x 10" cells/ml). « , 1 mg/1; A, 2 mg/1; •,5mg/l; • , 10mg/l.

Table HL Inhibitory activity of tazobactam, davulanic acid and sulbactam against various types of /Mactamase.

Strains Esch. coli ML4901/Rms212 Stapk aureus ML15009/pI258 K. pneumoniae GN69 Prot vulgaris GN7919 Esch. coli GN5482 C. freundli GN7391 Eta. cloacae GN7471 Ser. marcticens GN10857 M. morganti GN5407 Pi. aeruginosa GN10362

Type of Type of 0-lactamase

PCaseType I»(TEM-1) PCaseType V* PCase» CXastf CSasef CSase1 CSase* CSase* CSaaef CSasf

ID5 tazobactam 0O02 0073 0-290 0041 4-83 0-74 10-1 3-00 0-147 0-768

clavulanate sulbactam 0O16 0-023 0-188 0088 >100 >100 >100 82-6 >100 >100

O270 0189 2-04 0257

104 3-94 9-83 2-69 0.588 1-49

TD jo was defined as the concentration giving 50% inhibition of /Mactamase after incubation for 5 min. 'Substrate penicillin G. 'Substrate: cephaJoridine.

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F. Higashitanl et al

was significantly inactivated by incubation with only 1 mg/1 of tazobactam and the inhibitory effect was maintained for 6h after initiation of the test, while 10 mg/1 of clavulanate or sulbactam were not as effective. Discussion

References Aronoff, S. C , Jacobs, M. R., Johenning, J. & Yamabe, S. (1984). Comparative activities of the /Mactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin. Antimicrobial Agents and Chemotherapy 26, 580-2. Brown, A. G., Butterworth, D., Cole, M., Hanscomb, G., Hood, J. D., Reading, C. et al. (1976). Naturally-occurring /Mactamase inhibitors with antibacterial activity. Journal of Antibiotics 29,668-9. English, A. R., Retsema, J. A., Girard, A. E., Lynch, J. E. & Barth, W. E. (1978). CP-45899, a beta-lactamase inhibitor that extends the antibacterial spectrum of beta-lactams: initial bacteriological characterization. Antimicrobial Agents and Chemotherapy 14, 414-9. Gutmann, L., Kitzis, M.-D., Yamabe, S. & Acar, J. F. (1986). Comparative evaluation of a new /Mactamase inhibitor, YTR 830, combined with different /Mactam antibiotics against bacteria harboring known /Mactamases. Antimicrobial Agents and Chemotherapy 29, 955-7. Jacobs, M. R., Aronoff, S. C , Johenning, S., Shlaes, D. M. & Yamabe, S. (1986). Comparative activities of the /Mactamase inhibitors YTR 830, clavulanate, and sulbactam combined with ampicillin and broad-spectrum penicillins against defined /Mactamase-producing aerobic gram-negative bacilli. Antimicrobial Agents and Chemotherapy 29, 980-5. Matsuura, M., Nakazawa, H., Hashimoto, T. & Mitsuhashi, S. (1980). Combined antibacterial activity of amoxicillin with clavulanic acid against ampicillin-resislant strains. Antimicrobial Agents and Chemotherapy 17, 908-11. Mitsuhashi, S. & Inoue, M. (1981). Mechanisms of resistance to /Mactam antibiotics. In Beta Lactam Antibiotics (Mitsuhashi, S., Ed.) pp. 41-56. Japan Scientific Societies Press, Tokyo. Paisley, J. W. & Washington, J. A. (1978). Combined activity of clavulanic acid and ticarcillin against ticarcillin-resistant, gram-negative bacilli. Antimicrobial Agents and Chemotherapy 14, 224-7. Reading, C. & Cole, M. (1977). Clavulanic acid: a beta-lactamase-inhibiting beta-lactam from Streptomyces davuligerus. Antimicrobial Agents and Chemotherapy 11, 852-7. (Received 19 September 1989; accepted 14 November 1989)

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The present in-vitro study was performed to evaluate the synergistic activity with piperacillin and /Mactamase inhibitory activity of tazobactam compared with other plactamase inhibitors. Our data indicate that tazobactam was an effective /Mactamase inhibitor, confirming the results of others (Aronoff et al., 1984; Gutmann et al., 1986; Jacobs et al., 1986). Piperacillin is a highly effective broad spectrum antibiotics, however it is labile to some /Mactamases. The combination of tazobactam with piperacillin showed a highly synergistic effect and could extend the spectrum of this antibiotic to include resistant /?-lactamase producing organisms. Tazobactam had potent inhibitory activity against plasmid-mediated /Mactamases and also some chromosomally-mediated enzymes such as those of K. pneumoniae, Prot. vtilgaris and M. morganii. On the basis of these observations, we conclude that tazobactam could overcome the problems caused by the /Mactamase lability of piperacillin, and piperacillin combined with tazobactam may be a promising combination for treatment of infections caused by both Gram-positive and Gram-negative bacteria.