Antibacterial activity of ticarcillin in the presence of clavulanate potassium

Antibacterial Activity of Ticarcillin in the Presence of Clavulanate Potassium

ROBERT SUTHERLAND, DSc. ANGELA S. BEALE, MI. Biol. RONALD J. BOON, BSc. KAREN E. GRIFFIN, MI. Biol. BRIAN SLOCOMBE, M.I. Biol. DEBRA H. STOKES, BSc. ANTHONY R. WHITE, B. Tech.

Betchworth, United Kingdom

From the Beecham Pharmaceuticals Research Division, Brockham Park, Betchworth, Surrey, RH3 7AJ, United Kingdom. Requests for reprints should be addressed to Dr. Robert Sutherland, Chemo- therapeutic Research Center, Brockham Park, Betchworth, Surrey, RH3 7AJ, United Kingdom.

Timentin and Augmentin are registered trademarks of Beecham Group p.1.c.

The antibacterial effects produced by ticarcillin disodium plus clavulanate potassium, a combination of the broad-spectrum penicillin ticarcillin, and the beta-lactamase inhibitor clavulanic acid as the potassium salt, have been measured in vitro and in experlmental infection studies. The presence of clavulanic acid resulted in a significant enhancement of the activity of ticarcillin against a wide range of beta-lactamase-producing bacteria. These included ticarcillin-resistant strains of Escherichia toll, Klebsiella pneumoniae, Proteus mhabilis, P. vulgaris, Yersinia enterocolltica, and the anaer- obe Bacteroides fragllls. In addition, beta-lactamase-producing lso- lates of Hemophilus lnfluenzae, Branhamella catarrhalis, Neisseria gonorrhoeae, and Staphylococcus aureus were susceptible to tlcarcillin and clavulanate. Clavulanic acid did not influence the activity of ticarcillln against ticarcillin-susceptible bacteria. The bactericidal effects of the antibiotic combination were measured in an in vitro kinetic model in which the drug concentrations were varied to simulate those measured in humans after intravenous dosing wlth ticarcillin (3.0 g) and clavulanate potassium (100 mg clavulanic acid). In these tests, tlcarcillin plus clavulanic acid had pronounced bactericidal activity against ticarcillin-resistant bacteria. The protection of ticarcillin by clavulanic acid from inactivation by bacterial beta-lactamases in vivo was demonstrated in experimental infection models in which the efficacy of the ticercillin plus clavulanic acid combination against infections caused by beta-lactamase-producing bacteria was correlated with the presence of effective concentrations of both antibiotic and Inhibitor at the slte of Infection.

Ticarcillin is a broad-spectrum penicillin active against a wide range of gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa, and is well established as a drug of choice in the treatment of infections due to susceptible bacteria. This penicillin shows moderate stabiiin-the-presenceof beta-lactamases of certain gram-negative bacilli producing the Richmond and Sykes Class I enzymes [l], e.g., Entero- batter species, indole-positive Proteus species, P. aeruginosa, Serratia marcescens, and also in the presence of enzymes produced by the anaerobic Bacteroides species. However, ticarcillin is unstable in the presence of most other bacterial beta-lactamases, inciuding the plasmid- mediated enzymes responsible for the resistance of increasing numbers of clinical isolates to beta-lactam antibiotics.

Clavulanic acid is a progressive inhibitor of the beta-lactamases’of staphylococci and most gram-negative bacteria [2,3] and is capable of protecting beta-lactam antibiotics from inactivation by these bacteria [4-

November 29, 1995 The Americen Journal of Medicine Volume 79 (ruppl5B) 13

SYMPOSIUM ON BETA-LACTAMASE INHIBITION-SUTHERLAND ET AL

Ticarcillin Disodium

oflz”” Clavulanate Potassium

fgure 1. Structures of ticarcillin disodium and clavulanate

1. The beta-lactamase inhibitory effects of clavulanic acid ave been demonstrated in vivo [7,8], and formulations of

amoxicillin plus clavulanic acid (Augmentin) have been shown to be effective in the treatment of a wide range of clinical infections caused by beta-lactamase-producing bacteria [Q-l 11.

This article describes experimental data demonstrating the antibacterial activity of ticarcillin in the presence of clavulanic acid, particularly against ticarcillin-resistant bacteria. Data from animal studies describing the penetration of ticarcillin and clavulanic acid to s/tes of infection and the efficacy of ticarcillin plus clavulanic acid against infections refractory to ticarcillin are also reported.

MATERIALS AND METHODS

Compounds. Ticarcillin and clavulanic acid were employed as ticarcillin disodium and clavulanate potassium in the studies reported and were laboratory reference standards sup- plied by Beecham Pharmaceuticals, United Kingdom Divi- sion, Worthing, Sussex, England (Flgure 1). Bacteria. Most of the bacterial strains tested were isolated from a variety of clinical sources. For comparison, standard laboratory strains were included in most in vitro tests. Antibacterial Ac+&y. Minimal inhibitory concentrations were determined by twofotd serial dilution into Mueller-Hinton agar, supplemented with defibrinated horse blood for tests with streptococci (5 percent volume per volume), B. catarrhalis, N. gonorrhoeae, N. meningitidis and Bacteroides species (10 percent volume per volume) and heated (chocolated) horse blood (10 percent volume per volume) for tests with H. influenzae and Gardnerella vaginalis. The surface of the agar was inoculated by means of a multi-point inoculator, which delivered 0.001 ml of an appropriate dilution of an overnight culture’ of the test organism to yield a final inoculum of lo4 cells per spot, except for tests with gram-positive cocci (5 x lo5 cells per spot). Minimal inhibitory concentration values were determined after 18 hours of incubation at 37% as

the lowest concentration that prevented visible microbial growth. In the case of anaerobic bacteria, the plates were incubated for a minimum of 24 hours. In these antibacterial tests, serial dilutions of ticarcillin were combined with 2 pg of clavulanic acid per ml as recommended by Fuchs et al [12]. This concentration of clavulanic acid is readily achieved and maintained in serum and blister fluid when a combination of ticarcillin plus clavulanic acid is administered to humans [13].

In the tests to measure the interaction of gentamicin with the beta-lactams, the activity of ticarcillin, ticarcillin plus clavulanic acid, and gentamicin alone and in combination were measured by an agar checkerboard test [14]. Bactericidal Activity. Minimal bactericidal concentrations were determined by serial dilution in 2.0 ml volumes of Muel- ler-Hinton broth, which were inoculated with an overnight broth culture diluted to yield an inoculum of approximately lo6 cells per ml. After 18 hours of incubation at 37°C a 0.01 ml volume from each tube showing no visible growth was trans- ferred onto antibiotic-free agar containing penicillinase and incubated at 37°C overnight. The minimal bactericidal concentration was defined as the lowest concentration at which the count was reduced to five colonies or less, a reduction of 99.9 percent of the bacterial inoculum.

The bactericidal activities of ticarcillin plus clavulanic acid and ticarcillin alone were compared in conventional time-kill curve tests in which Mueller-Hinton broth (20 ml) containing selected concentrations of the compounds was inoculated with an overnight broth culture of a test strain of Klebsiella pneumoniae. The flasks were shaken in an orbital incubator at 37”C, samples were taken at intervals, and 0.05 ml volumes of suitable dilutions were pipetted onto antibiotic-free agar plates. Bacterial colonies were counted after overnight incubation at 37% and the viable counts calculated.

The effect of clavulanic acid on the bactericidal activity of ticarcillin against a ticarcillin-resistant strain of K. pneumoniae Ba95 (ticarcillin minimal inhibitory concentration greater than 512 pg/ml) was studied in an in vitro kinetic model in which the concentrations of antibiotic and inhibitor were varied to simulate the serum concentrations achieved in humans after intravenous administration of 3.0 g of ticarcillin plus 100 mg of clavulanic acid (Timentin). The in vitro system employed to simulate the one-compartment pharmacokinet- its of an intravenous infusion was essentially that described by Grass0 et al [15]. A multi-channel peristaltic pump con- veyed sterile antibiotic-free Mueller-Hinton broth from a res- ervoir, at a set-flow rate, to a flask containing a predeter- mined concentration of antibiotic in broth. The antibiotic was pumped at the same rate to another flask, which contained an actively growing culture of the test orQanism.The flow rates in the model were calculated to give a maximum concentration and rate of elimination similar to those observed in humans [13,16]. Experimental Infections. Mice were infected by the intraperitoneal route with 0.5 ml of a suspension in hog gastric mucin of a dilution of an overnight broth culture of the test organism standardized to an infective inoculum of IO to 100 median lethal doses. The compounds were administered by the subcutaneous route one and five hours or one, three, five, and seven hours after infection. The drugs were given at four dose levels for each test and groups of 10 mice were treated

14 November 29, la85 The Amerkan Journal of Medklne Volume 79 (suppl5B)

at each dose level. The numbers of animals surviving for four days after infection were recorded and the dose of drug re- quired to produce protection of 50 percent of infected animals was calculated. The concentrations of ticarcillin and clavulanic acid present in peritoneal washings from mice infected with a beta-lactamase-producing strain of P. aeruginosa were measured after administration of the first dose at one hour after infection. Samples were collected from groups of five mice at each time interval, pooled, and assayed for ticarcillin content by the agar diffusion assay with P. aeruginosa ATCC 29336 and for clavulanic acid by a beta-lactamase inhibition microbiological assay with K. pneumoniae ATCC 29665 [7j.

The effects of therapy in a localized wound lesion were determined in an experimental model in which mice were infected with 0.5 ml of a 22-hour culture of 8. fragilis VPI 6908, 2 x 10’ cells, in semi-solid agar by subcutaneous injection in the groin. Subcutaneous therapy with ticarcillin or ticarcillin plus clavulanic acid was started at the time of infection and continued three times a day for four days. Groups of 10 mice were killed seven days after infection, and the bacterial numbers in homogenates prepared from groin tissue were counted [7]. The drug concentrations in the pus of fully formed abscesses were also measured after subcutaneous administration of ticarcillin or ticarcillin plus clavulanic acid.

RESULTS

Antibacterial Spectrum. The minimal inhibitory concentrations of ticarcillin plus clavulanic acid (ticarcillin plus 2 pg of clavulanic acid per ml) against representative strains of gram-positive and gram-negative bacteria are compared with those of ticarcillin and clavulanic acid in Tables I, II, and Ill. In general, clavulanic acid showed only a moderate level of antibacterial activity against most bacteria and, as a consequence, ticarcillin-susceptible strains of bacteria were usually no more susceptible to ticarcillin plus clavulanic acid than to ticarcillin alone.

In contrast, a high proportion of the beta-lactamase- producing strains of bacteria tested were susceptible to ticarcillin in the presence of clavulanic acid as a result of the beta-lactamase inhibitory effects of the latter drug. These included the beta-lactamase-producing strains of Staphylococcus aureus, S. epidermidis, B. catarrhalis, H. influenzae, and N. gonorrhoeae. The methicillin-resistant strain of S. aureus was more susceptible to ticarcillin in the presence of clavulanic acid, but remained relatively resistant to the combination as a result of the non-beta- lactamase-mediated resistance of this organism. Like- wise, ticarcillin and clavulanic acid showed a high level of activity against many of the ticarcillin-resistant strains of Enterobacteriaceae, including Escherichia coli, Klebsiella species, P. mirabilis, P. vulgaris, and Yersinia enterocolitica. In addition, the anaerobic gram-negative bacilli were notably more susceptible to ticarcillin plus clavulanic acid than to ticarcillin alone, with the beta-lactamases produced by Bacteroides species being very susceptible to inhibition by clavulanic acid.

However, ticarcillin-resistant bacteria producing the


Richmond and Sykes Class I beta-lactamases (cephalo- sporinases) were usually resistant to the combination of ticarcillin and clavulanic acid, which reflected the poor inhibitory activity of clavulanic acid against these enzymes [2,3]. These bacteria included Acinetobacter species, Citrobacter species, Enterobacter species, Morganella morganii, Providencia rettgeri, Pseudomonas species, including P. aeruginosa, and S. marcescens. Activity against Clinical Isolates. The results in Table IV show the concentrations of ticarcillin plus clavulanic acid and ticarcillin inhibiting 75 percent (MI&) and 90 percent (MI&,) of 1,298 clinical isolates of gram-negative bacteria and staphylococci. Ticarcillin plus clavulanic acid was active at relatively low concentrations against most strains of E. coli, P. mirabilis, P. stuartii, Y. enterocolitica, Bacteroides species, H. influenzae, and S. aureus. The reaction of K. pneumoniae varied, with the wild-type strains possessing chromosomally mediated beta-lactamases being highly susceptible to ticarcillin in the presence of clavulanic acid and the strains possessing plasmid-mediated beta-lactamases being more resistant to the combination. Ticarcillin plus clavulanic acid was notably more active than ticarcillin against all these species. In contrast, the MI& and MIC& values for ticarcillin plus clavulanic acid against the isolates of E. cloacae, S. marcescens, and P. aeruginosa were relatively high and not very different from those of ticarcillin alone.

The comparative activities of ticarcillin plus clavulanic acid and the broad-spectrum penicillins ampicillin, ticarcillin, mezlocillin, and piperacillin against beta-lactamase- producing isolates are illustrated in Figure 2. Against E. coli, K. pneumoniae, and Proteus mirabilis, ticarcillin plus clavulanic acid was the most active agent, followed in descending order of activity by mezlocillin and piperacillin and by ticarcillin and ampicillin. Against S. marcescens, ticarcillin plus clavulanic acid, mezlocillin, and piperacillin showed similar activities and were notably more active than ampicillin and ticarcillin. The beta-lactamase-producing isolates of S. aureus were highly resistant to ampicillin and to the ureidopenicillins mezlocillin and piperacillin as a result of the instability of these compounds to staphylo- coccal beta-lactamase; ticarcillin was rather more active and ticarcillin plus clavulanic acid displayed a uniform high level of activity (minimal inhibitory concentration range: 4.0 to 8.0 pg/ml). Factors Affecting Antibacterial Activity. In general, the activity of ticarcillin plus clavulanic acid was unaf- fected by the composition of the more common bacterio- logical media. For instance, the minimal inhibitory concentration values of the combination were similar in Mueller-Hinton agar and broth (Difco), Diagnostic Sensi- tivity Test agar (Oxoid), Blood Agar Base (Oxoid), nutrient agar and nutrient broth (Oxoid), or Tryptone Soya broth (Oxoid). Similarly, the activity of ticarcillin plus clavulanic acid was not greatly influenced by the pH of the test me-

November 29, 196!5 The American Journal of Medicine Volume 79 (wppl 58) 15

TABLE I Antibacterial Spectrum of Ticarcillin plus Clavulanic Acid, Ticercillin, and Clavulanic Acid against Aerobic Gram-Positive Bacteria

Orpanlsm Tlcarcillin plus Clavulanlc Acid* Tlcarcillln Clavulanlc Acid

Staphylococcus aureus ATCC 25923 Staphylococcus aureus NCTC 11561t Staphylococcus aureus 1517* Staphylococcus epidermidis 80/298 Staphylococcus saprophyticus 20 Streptococcus pyogenes 421 Streptococcus agalactiae 2866 Streptococcus pneumoniae 1760 Streptococcus faecalis ATCC 29212 Listeria monocytogenes 5248 Nocardia asteroides 1867

MC = minimal inhibitory concentration. ‘Ticarcillin plus 2 pg of clavulanic acid per ml. +Beta-lactamase-producing strain. *Methicillin-resistant strain.

0.5 1.0 16 1.0 64 16

64 128 512 4.0 128 64 1.0 1.0 16 0.1 0.25 64 2.0 2.0 64 1.0 1.0 64

64 64 512 4.0 2.0 32 8.0 4.0 128

TABLE II Antibacterial Spectrum of Ticarcillin plus Clavulanic Acid, Ticarcillln, and Clavulanic ACM against Aerobic Gram-Negatlve Bacteria

Orpanlsm Tlcarclllln plus Clavulanlc Acid’ Tlcarclllln Clavulanlc Acid

Branhamella catarrhalis 1502 0.06 0.5 4.0 Branhamella catarrhalis 2001 E 0.12 64 4.0 Hemophilus influenzae 3037 0.25 0.25 128 Hemophilus influenzae 2406’ 0.25 128 128 Neisseria meningitidis 2054 10.01 0.06 2.0 Neisseria gonorrhoeae 144 co.01 0.25 2.0 Neisseria gonorrhoeae 1584+ 1.0 64 4.0 Gardnerella vaginalis 879 0.25 0.5 32 Yersinia enterocolitica 10688 0.25 32 32 Escherichia coli ATCC 25922 4.0 4.0 16 Escherichia coli ATCC 35218+ 8.0 >512 16 Klebsiella pneumoniae 112 4.0 128 32 Klebsiella oxytoca Du 192 128 >512 32 Klebsiella ozaenae N141 2.0 128 32 Klebsiella rhinoscleromatis NCTC 5051 0.5 64 32 Proteus mirabilis 977 0.5 0.5 64 Proteus mirabilis 899 8.0 >512 32 Proteus vulgaris 849 0.5 2.0 128 Proteus vulgarts 276 2.0 256 128 Citrobacter freundii 48034 2.0 2.0 32 Enterobacter aerogenes T218 4.0 4.0 64 Enterobacter cloacae T748 2.0 4.0 32 Morganella morganii F 8.0 16 128 Providencia rettgeri B 2.0 2.0 64 Providencia stuartii 21057 0.5 0.5 64 Serratia marcescens US32 2.0 2.0 128 Pseudomonas aeruginosa ATCC 27853 8.0 8.0 128 Pseudomonas fluorescens D 128 512 128 Pseudomonas putida 5516 64 64 128 Pseudomonas cepacia 1054 64 64 128 Pseudomonas maltophilia 1120 64 128 32 Acinetobacter anttratus WIG1 128 >512 16 Aeromonas hydrophila US3 32 64 16 Flavobacterium meningosepticum 10580 16 16 128

MC = minimal inhibitory concentration. ‘Ticarcillin plus 2 w of clavulanic acid per ml. +Ticarcillin-resistant strain.

16 November 29,1966 The American Journal of M~lclnr Volume 79 (auppl6B)


TABLE III Antibacterial Spectrum of Ticarcillin plus Clavulanic Acid, Ticarcillin, and Clavulanic Acid against Anaerobic Bacteria

MIC (fro/ml)

Organism Tlcarclllln plus Clavulanlc Acid’ Ticarclllln Clavulanlc Acid

Clostridium difficile 17624 32 32 >512 Clostridium tertium 2217 16 16 32 Peptococcus prevotii 3725 <O.l 0.25 2.0 Peptostreptococcus anaerobius 1013 1 .o 1.0 32 Bacteroides fragilis 83 1.0 16 16 Bacteroides fragilis BC16 6.0 512 16 Bacteroides distasonis 179 6.0 32 32 Bacteroides ovatus 11361 4.0 126 32 Bacteroides vulgatus 11341 6.0 512 16 Bacteroides bivius 6041 2.0 64 16 Bacteroides melaninogenicus 7342 0.5 64 16 Bacteroides asaccharolyticus 10963 6.0 512 64 Fusobacterium necrophorum 7935 co.01 0.03 1 .o Fusobacterium nucleatum NCTC 10562 10.01 0.1 0.5 Fusobacterium varium NCTC 10560 1.0 2.0 32

MC = minimal inhibitory concentration. ‘Ticarcillin plus 2 pg of clavulanic acid per ml.

Figure 2. Comparative activities of ticarcillin plus clavulanic acid, ticarcillin, ampicillin, mezlocillin, and piperacillin against beta-lactamase-producing isolates.

Escherichi di (20) Kbbsiella p~umoniac (19)

60

0 2.0 4.0 6’0 16 32 64 128 256 512 5512 concentration @g/ml)

n ‘y a Proteus mirabils (20)

s 20 F

1’0 2’0 4.0 80 16 32 64 128 256 512 a512

3 concentration (MS/ml)

staphyklcoccus aweUs (15) loo- / - 60.

60.

40. .[I L

21L-.-- 1.0 2’0 4.0 8’0 16 32 64 128256512 ,512

concentration (pg/ml)

di!?i?! _ ’ / 1.0 2.0 4.0 8’0 16 32 64 128 256 512 ~512

concentration (pg/ml)

Senatia mavxscens (19)

I . 1

l’0 PO 4.0 6’0 16 32 64 128 256 512 >512 concentration (pglml)

l Ticarcillin/Clavulan~c Acid 0 Tlcarcilhn - Amp!cllhn c Mezloc~llm L. Plperacilhn

November 29,1985 The American Journal of Medlclne Volume 79 (ruppl5B) 17


TABLE IV Activity of Ticarciiiin plus Ciavuianic Acid and Ticarciiiin against Clinical isolates

Organism (number) Antibiotic

MC Range WW

Escherichia coli (348)

Klebsiella pneumoniae (70)

Klebsiella pneumoniae (W

Proteus mirabilis l

(152) Proteus vulgaris

(27) Providencia stuartii

(24) Enterobacter cloacae

(52) Serratia marcescens

(57) Pseudomonas aeruginosa

VW Yersinia enterocolitica

(16) Bacteroides species

(38) Hemophilus influenzae

(28) Staphylococcus aureus

(238)

Ticarcillin plus clavulanic acid* Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin Ticarcillin plus clavulanic acid Ticarcillin

0.5-128 8.0 32 0.5->512 >512 ~512 1 .O-32 4.0 4.0 8.0-258 258 258 2.0-258 84 128

512->512 >512 >512 0.5-84 1.0 8.0 0.5->512 2.0 512 0.1-84 2.0 8.0 O.l->512 18 258 0.5-258 8.0 18 0.5-r512 512 Ml2 l.O-~512 84 512 2.0-~512 128 >512 1.0-512 84 128 1 .O->512 >512 >512 8.0->512 128 512 4.0->512 512 >512

0.25-4.0 0.5 2.0 18-X4 84 X4

0.25-18 1.0 4.0 8.0-512 84 512

0.25-0.5 0.5 0.5 0.25-X4 >84 S-84

0.5-4.0 4.0 4.0 0.5-128 18 32

MC = minimal inhibitory concentration: MICrs and MC w = minimal inhibitory concentrations resulting in 75 and 90 percent inhibition, respec- tively. ‘Tlcarcillin plus 2 cog of clavulanic acid per ml.

dium, and the minimal inhibitory concentrations were gen- erally similar in media buffered to pH 6.0, pH 7.0, or pH 8.0. The antibacterial activity of ticarciiiin plus ciavuianic acid was not significantly influenced by the presence of human serum, as neither ticarciiiin (50 percent bound) nor clavuianic acid (25 percent bound) is highly bound to the protein of human serum.

The effect of changes in ,inocuium size on the activity of ticarciilin plus ciavuianic acid is illustrated in Table V. The results show that the minimal inhibitory concentrations of the combination increased only twofold to fourfold as the size of the inoculum increased from 1 O2 cells to 10’ cells in the case of the ticarcillin-resistant strains of E. coii JT39 and K. pneumoniae R112. Similarly, the activity of ticarciiiin and ciavuianic acid against the beta-iactamase-producing strain of S. aureus NCTC 11561 was virtually un- changed as the inocuium size was varied from 10 ceils to lo5 cells, whereas the activity of ticarciliin decreased thirty-twofold as a result of the beta-iactamase activity of the larger inocuium. Bactericidal Activity. The minimal bactericidal concentrations of ticarciiiin plus ciavuianic acid were the same as or twofold higher than the minimal inhibitory concentrations against the test bacteria, including the beta-iac-

tamase-producing strains. The combination was more effective in these tests than was ticarciiiin alone (Table VI).

The bactericidal effects produced by ticarciiiin plus ciavuianic acid and by ticarciliin in conventional time-kill curve tests against two strains of K. pneumoniae R112 (ticarciiiin minimal inhibitory concentration equal to 128 &ml) and K. pneumoniae Ba95 (ticarciiiin minimal inhibitory concentration greater than 512 N/ml) are illustrated in Figure 3. Against K. pneumoniae R112, ticarciiiin (64 pg/mi) caused an initial bactericidal effect, reducing the inocuium from 2 x 1 O6 ceils per ml to 2 x 1 O5 ceils per ml at four and 8 hours. The numbers increased to 3 x IO’ ceils per ml at 24 hours, at which time the concentration of ticarciiiin had decreased from 64 to 10 &ml. in contrast, in the presence of ciavuianic acid, ticarciiiin (16 &ml) caused a 99.9 percent reduction in bacterial ceils by 8 hours, as a result of the inhibition of the Kiebsieiia beta- iactamase by ciavulanic acid, which protected ticarciiiin during the test. in the case of the more resistant strain, K. pneumoniae Ba95, ticarciiiin (256 &ml) was com- pletely ineffective, with the ceil numbers increasing. at the same rate as those in the untreated control culture, and no ticarciiiin was detectable at the end of the test. On the other hand, ticarciiiin (64 M/ml) plus ciavuianic acid was

18 Nowmber 29,198!5 The Amerkan Journal ol MedIche Volume 79 (suppl5B)


TABLE V Effect of lnoculum Size on the Activity of Ticarcillin plus Clavulanic Acid and Ticarclllin

Organism lnoculum

(Colony-forming units)

MIC Wml) Ticarciilln plus Ciavulanlc Acid’ Ticarciilin

Escherichia coli NCTC 10418 106 2.0 2.0 104 2.0 2.0 102 1 .o 2.0

Escherichia coli JT39 lo6 32 >512 lo4 16 ,512 ld 8 >512

Klebsiella pneumoniae R112 106 2.0 256 104 2.0 128 ld 1.0 64

Staphylococcus aureus NCTC 11561 105 4.0 128 103 2.0 8.0 10 2.0 4.0

MIC = minimal inhibitory concentration. “Ticarcillin plus 2 w of clavulanic acid per ml.

TABLE VI Minimal Bactericidal Concentrations of Tlcarcillin plus Clavulanic Acid and Tlcarclllln

Ornanlsm

Ticaniliin plus Clavulanic Acid’

MIC MBC

Ticarslliln

MC MQC

Escherichia coli ATCC 25922 4.0 8.0 8.0 Escherichia coli JT39 32 32 >512 Ktebsiella pneumoniae R112 2.0 4.0 128 Pseudomonas aeruginosa ATCC 27853 32 64 32 Serratia marcescens Sm52 64 64 >512 Proteus vulgaris X 2.0 2.0 16 Staphylococcus aureus NCTC 11561 2.0 4.0 16

MIC = minimal inhibitory concentration in w/ml; MBC = minimal bactericidal concentration in &ml. l Ticarcillin plus 2 pg of clavulanic acid per ml.

8.0 >512

256 256

>512 512 256

K. pneumoniae R112 K. pneumoniae Ba 95

-a m (13)

102 -

4 8 12 ‘4

Figure 3. Bactericidal activities of ticarcillin plus clavulanic acid and ticarcillin against Klebsiella pneumoniae in time-kill curve tests. Left, A, untreated control; 0, ticarciiiin (64 l@ml); 0, ticarcillin (16 pglml) plus clavulanic acid (2 pglml). Right, A, untreated control; 0, ticarcillin (256 ~glml); l , ticarcillin (64 pglml) plus clavulanic acid (2 rglml). Numbers in parentheses are concentrations (pgiml) measured. hours hours

November 2S,lSS5 The Amsrlcan Journal of Medicine Volume 79 (suppl5B) 1S


I l T~carc~llm (human) T,mnn+,n 7 I" A T~carc~llm (m wtro)

I Clavulamc Acid (human) 1 0 Clavulanlc Acid (m vitro)

Tlcarclllm 3.09 A Ttcarcillin (in v&o)

hours b.

109

108 Bacterial 1~7

(cEE$106

Q

A Ticarcillin (3.09) double dose lJTimentin (3’19) single dose WTlmentin (3%) double dose

105 n

104 103 1021 8 8 a a ’ ’ 1 2 3 4 5 6 7’74

hours

bactericidal, and there was no evidence of inactivation of ticarcillin by the organism during the test.

The bactericidal effects produced by ticarcillin plus clavulanic acid against the ticarcillin-resistant strain of K. pneumoniae Ba95 in the in vitro kinetic model, in which the antibiotic concentrations were varied to simulate those obtained in humans, are illustrated in Figure 4. The culture was exposed to concentrations of ticarcillin and clavulanic acid similar to those measured in human subjects after a 30-minute infusion of 3.1 g of Timentin (3.0 g of ticarcillin plus 100 mg of clavulanic acid) (Figure 4a) [16]. The figure also shows the ticarcillin concentrations of a simulated 3.0 g dose of ticarcillin; there was no evidence of overt inactivation of the penicillin by K. pneumoniae Ba95 in the culture flask. Nevertheless, the simulated dose of ticarcillin caused no reduction in the number of bacteria, with the bacterial growth being similar to that of the untreated control culture (Figure 4b). In contrast, ticarcillin plus clavulanic acid demonstrated rapid bactericidal activity, causing more than a 99 percent reduction in bac-

Figure 4. Bactericidal effects of simulated infusion of ticarcillin (3.0 g) plus clavulanic acid (100 mg) and ticarcillin (3.0 g) against Klebsiella pneumoniae Ba95.

terial cells within four hours. A simulated second dose at four hours did not cause a further reduction in bacterial numbers, but did result in a prolongation of the antibacterial activity of the formulation. That is, in the culture exposed to a single simulated dose of Timentin, there was a small increase in bacterial numbers at seven hours as the antibiotic and inhibitor were eliminated from the system, and at 24 hours the bacterial counts were similar to those of the control culture. In the culture receiving a second simulated dose of Timentin, the extent of growth at 24 hours was notably reduced. Selection of Resistant Variants. The rate of emergence of resistant variants was determined by repeated subculture of the test strains of E. coli, P. aeruginosa, E. cloacae, and S. aureus in broth containing ticarcillin plus clavulanic acid, ticarcillin, or piperacillin (Table VII). In general, bacterial resistance developed to all three antibiotics in ‘a gradual stepwise fashion characteristic of beta-lactam antibiotics, and there was no evidence of the rapid emergence of resistant bacteria. There was no dif-

20 November 29,1@85 The American Journal of Medicine Volume 79 (suppl5B)

SYMPOSIUM ON BETA-I-ACTAMASE INHIBITION-SUTHERLAND ET AL

TABLE VII Selection of Resistant Variants during Repeated Subculture in the Presence of Ticarciiiin plus Ciavuianic Acid, Ticarciliin, and Piperaciilin

MIC (au/ml) and Subcultura Number

Organism Antibiotic 0 1 2 3 4 5 6 7

Escherichia coli ATCC 25922

Pseudomonas aeruginosa ATCC 27853

Enterobacter cloacae T629

Staphylococcus aureus ATCC 25923

Ticarcillin plus clavulanic acid’ 4 8 16 16 16 32 32 64 Ticarcillin 4 8 16 16 16 64 64 64 Piperacillin 2 2 a 16 32 64 64 128 Ticarcillin plus clavulanic acid 64 128 256 256 512 512 1024 1024 Ticarcillin 32 64 128 256 512 1024 1024 2048 Piperacillin 16 64 256 1024 512 1024 1024 1024 Ticarcillin plus clavulanic acid 8 32 128 128 1024 1024 1024 2048 Ticarcillin 4 32 64 512 512 1024 2048 4096 Piperacillin 2 8 256 1024 1024 1024 1024 1024 Ticarcillin plus clavulanic acid 0.5 0.5 1 1 1 2 2 2 Ticarcillin 0.5 0.5 1 2 1 2 1 2 Piperacillin 0.25 0.5 0.5 0.5 0.5 0.5 0.25 1

MIC = minimal inhibitory concentration. l Ticarcillin plus 2 pg of clavulanic acid per ml.

TABLE VIII Synergistic Activity of Ticarcillin plus Clavulanic Acid Combined with Gentamicin

MIC (pplml)

Antlblotic Pseudomonas aeruginosa

ATCC 21553 Pseudomonas aeruginosa

Dalglelsh Serratla marcascens

3976

Ticarcillin 16 >512 >512 Ticarcillin plus clavulanic acid 16 128 64 Gentamicin 2 2 16 Ticarcillin plus gentamicin 4 + 0.5 -t -t

Ticarcillin, clavulanic acid’ plus gentamicin 4 + 0.5 32 + 0.25 16+4

MC = minimal inhibitory concentration. ‘Ticarcillin plus 2 pg of clavulanic acid per ml. tNo synergy detected; activity of gentamicin only.

ference between ticarcillin plus ciavulanic acid and ticarcillin alone with respect to the rate of selection of resistant variants in these tests. Interaction with Gentamicin. The results of checkerboard tests to measure the antibacterial effects produced by combining ticarcillin and clavulanic acid with gentami- tin are shown in Table VIII. The data show that ticarcillin plus clavulanic acid and ticarcillin alone produced similar synergistic effects in combination with gentamicin against the ticarcillin- and gentamicin-susceptible strain of P. aeruginosa ATCC 27853. Against the ticarcillin-resistant (beta-lactamase-producing) strain of P. aeruginosa Dalgleish, there was no synergy between ticarcillin and gentamicin as a result of inactivation of ticarcillin by the organism, but synergistic effects were obtained with ticarcillin plus clavulanic acid plus gentamicin, which reflected the beta-lactamase inhibitory effects of clavulanic acid. Likewise, synergistic effects were observed between ticarcillin plus clavulanic acid and gentamicin against the ticarcillin-resistant, gentamicin-resistant strain of S. mar-

cescens 3976, whereas synergy was not observed between ticarciliin and gentamicin. Activity against Experimental Infections. The effects of ticarcillin plus clavulanic acid and ticarcillin alone in the treatment of bacteremic infections in mice caused by ticarcillin-resistant bacteria are illustrated in Figure 5 and Table IX. The data in Figure 5 show the concentrations of ticarcillin and clavulanic acid measured in the peritoneal washings from mice infected by the intraperitoneal route with a beta-lactamase-producing strain of P. aeruginosa. The subcutaneous dose employed (375 ‘mglkg of ticarcillin plus 12.5 mg/kg of clavulanic acid) resulted in concentrations of penicillin and inhibitor of the same order as those measured in humans after administration of a 3.1 g dose of Timentin (Figure 4), but the rate of elimination of these compounds from mice was more rapid than in humans. The results of treating the infections produced by intraperitoneal inoculation of the infecting bacteria are shown in Table IX. Ticarcillin plus clavulanic acid was effi- cacious against the infections caused by ticarcillin-resis-

November 29, 1995 The American Journal of Medicine Volume 79 (suppl 58) 21


l Ticarcillin 375mg/kg +

A Clavulanic aad 125mglkg

0 Ticarcillin 375mglkg

Clavulanic

minutes

Figure 5. Concentrations of ticarcillin and clavulanic acid in peritoneal washings of mice infected with Pseudomonas aeruginosa Dalgleish.

tant strains of E. coli, P. mirabilis, K. pneumoniae, P. aeruginosa, and S. aureus, all of which were refractory to treatment with ticarcillin.

The effects of treating a localized lesion infection in mice caused by B. fragilis VPI 8908 are shown in Table X and Figure 6. The concentrations of ticarcillin and clavulanic acid measured in pus aspirated from abscesses seven days after infection with B. fragilis are shown in Table X and were concentrations achievable in tissue fluid of humans [13]. Pustular abscesses developed seven days after infection in all untreated animals, and bacterial counts of 10’ to 10’ bacteria per g of tissue were meas-

ured (Figure 6). Therapy with either ticarcillin or clavulanic acid was largely ineffective in reducing the number of abscesses formed or viable bacteria recovered. In contrast, ticarcillin plus clavulanic acid caused a significant reduction in both the number of bacteria recovered and groin abscesses formed.

COMMENTS

The results reported herein show that the beta-lactamase inhibitor clavulanic acid is capable of protecting ticarcillin from inactivation by bacterial beta-lactamases in antibacterial in vitro tests and in experimental infections. As a consequence, ticarcillin demonstrated significant activity in the presence of clavulanic acid against a wide range of ticarcillin-susceptible and ticarcillin-resistant bacteria. The latter group included beta-lactamase-producing strains of E. coli, K. pneumoniae, P. mirabilis, P. vulgaris, P. stuartii, Y. enterocolitica, and the anaerobic Bacteroides species. In addition, beta-lactamase-producing strains of B. catarrhalis, H. influenzae, and N. gonorrhoeae, all resistant to ticarcillin, were highly susceptible to ticarcillin plus clavulanic acid. Similarly, beta-lactamase-producing strains of S. aureus and S. epidermidis were inhibited by ticarcillin in the presence of clavulanic acid. However, methicillin-resistant strains were relatively insusceptible to the combination.

Most ticarcillin-resistant strains of Acinetobacter species, Enterobacter species, P. aeruginosa, and S. marcescens possessing Class I enzymes resistant to inhibition by clavulanic acid remained resistant to ticarcillin plus clavulanic acid. A number of ticarcillin-resistant isolates of these species were relatively susceptible to the combina-

TABLE IX Efficacy of Tlcarcillin plus Clavulanic Acid against lntraparitonaal Infections in Mica Refractory to Tlcarclllln*

Organism

Number of Ticarcillin plus Doses Clavulanic Acid

(subcutaneous) OWht) PWCEllt

Protection

Escherichia coli JT39

Proteus mirabilis C889

Klebsiella pneumoniae T76Z

Pseudomonas aeruginosa Dalgleish

Staphylococcus aureus MS9

2 187.5 + 6.25 100 93.8 + 3.12 90 46.9 + 1.56 60

2 375 + 12.5 100 187.5 + 6.25 20

93.8 + 3.12 20 46.9 + 1.56 0

2 187.5 + 8.25 100 93.8 + 3.12 90 46.9 + 1.56 30

4 750+25 100 375 f 12.5 90

187.5 + 6.25 40 93.8 + 3.12 0

2 46.9 + 1.56 100 23.5 + 0.78 60 11.8 + 0.39 40

~icarcillin was ineffective at the highest doses employed.

22 November 2Q,1965 The Americen Journal of Mediclne Volume 79 (suppl5B)

TABLE X Concentrations of Ticarcillin and Clavulanic Acid in Pus from Mice Infected with Bacteroides fragilis VPI 8888

Thenpy Dose

OwMt)

Concentration (rg/ml)

15 30 00 90 120 180 240 (minutes)

Ticarcillin plus clavulanic acid 900+30 37 71.7 90.9 67.6 N/S N/S 26.2 3.9 4.0 4.0 2.6 1.2 0.7 co.6

Ticarcillin 900 72.6 74.2 56.6 36.7 N/S N/S 5.4 N/S = no sample.

Figure 8. Effects of treatment with ticarcillin plus clavulanic acid (TIC + C.A.), ticarcillin (TIC), and clavulanic acid (C.A.) against Bacteroides fragilis VP/ 8908 groin infection in mice.

3 log- z ‘s 108 - i g/g

P 107 - --is-

.z 106 - 0

5 0 bacterial count from

CI, 106- 0” one mouse

b 104- -geometric mean count

8 103 - -@- @ ( ) % animals with abscesses

cm

lo2 T0C.A. 0

TIC Controls mg/kg (900 +30) (900) I

tion, which may have resulted from the inhibition of plasmid-mediated beta-lactamases by the inhibitor. The find- ings of these, antibacterial susceptibility tests were in keeping with those reported by other investigators [6,17- 201.

The compatability of ticarcillin and clavulanic acid was demonstrated in the bactericidal tests in which the concentrations of antibiotic and inhibitor were varied to simulate those measured in human subjects after administration of the ticarcillin plus clavulanic acid formulation. The simulated 3.1 g dose of Timentin had pronounced bactericidal effects against the highly resistant K. pneumoniae Ba95 strain. A simulated 3.0 g dose of ticarcillin was totally ineffective against this same strain.

The synergistic activity of combinations of ticarcillin and clavulanic acid plus gentamicin against ticarcillin-resistant bacteria not responding to combined treatment with ticarcillin plus gentamicin is of obvious interest. The synergistic effects produced by combining ticarcillin with aminogly- cosides are well established but, in a number of studies, synergy was not observed in tests against resistant bacteria, presumably because of rapid inactivation of the penicillin [*l-23]. Protection of ticarcillin by clavulanic acid apparently allows the synergistic interaction between ti-

carcillin and gentamicin to take place. Synergistic effects similar to those reported herein have also been observed in tests with ticarcillin and clavulanic acid plus amikacin or tobramycin (unpublished data).

The antibacterial activity of ticarcillin plus clavulanic acid against beta-lactamase-producing bacteria in vitro was confirmed by the efficacy of the combination against experimental infections refractory to ticarcillin. The results of the tests reported showed that both substances pene- trated to the sites of infection studied. At concentrations achievable in humans, the combination was effective against infections caused by beta-lactamase-producing strains of bacteria resistant to ticarcillin, which demonstrated the beta-lactamase inhibitory effects of clavulanic acid in vivo. Overall, the results of the antibacterial in vitro tests and the experimental infections reported herein illus- trate the therapeutic potential of ticarcillin in the presence of clavulanic acid.

ACKNOWLEDGMENT

We wish to thank Nigel Eaton, Anne Exell, John Gisby, and Jennifer Tyler for skilled technical assistance, Steve Lomax for preparing the illustrations, and Liz Clarke for typing the manuscript.

November 29,1995 The American Journal of Medicine Volume 79 (suppl se) 23


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24 November 29,1985 The American Journal of Medlcine Volume 79 (ruppl5B)

Antibacterial activity of ticarcillin in the presence of clavulanate potassium

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