DIAGN MICROBIOL INFECT DIS 7 1985;3:7-17

Apalcillin (PC-904): Spectrum of Activity and -Lactamase Hydrolysis/Inhibition

Arthur L. Barry, Ronald N. Jones, and Clyde Thornsberry

Apalci l l in is a Pseudomonas-active penici l l in with a broad spectrum of antibacterial activity similar to that of piperacillin, except for the greater potency of apalcillin against Acinetobacter spp. and Pseudomonas aeruginosa. Studies with 846 isolates representative of the common bacterial pathogens compared apalcillin to piperucillin, azlocillin, mezlocillin, carbenicillin, ticarcillin, cefotaxime, and cefoperuzone. Apalcillin and piperacillin were both active against all 13 species of the Enterobacteriaceae that were tested (MIC 5Os ~< 8.0 ~g/ml)but some strains were resistant to both penicillins. Apalcillin was active against Pseudomonas aeruginosa and Acinetobacter spp. (MIC 50 < 2.0 ?tg/ml). Like other penicillins, apalcillin was not effective against fl-lactamase-producing Staphylococcus spp., Haemophilus spp. or Neisseria gonor- rhoeae. Rates of hydrolysis by six ~-lactamase preparations from gram-negative bacilli were determined, comparing apalcillin, piperacillin, azlocillin, ticarcillin, ampicillin and diclox- acillin to benzyl penicilin. Apalciliin and ticarci|lin were more resistent than piperacillin and azlocillin to hydrolysis by the PSE-2 enzyme from P. aeruginosa. As did many other penicillins, apalcillin inhibited the Type I [3-1actamase that is produced by Enterobacter cloacae. The other enzymes were inhibitory only in very high concentrations.

INTRODUCTION

Apalcillin is a naphthydr idine derivative of ampicillin. It:has a broad spectrum of antibacterial activity, including activity against Pseudomonas aeruginosa (Noguchi et al., 1976; Bodey et al., 1978; Noguchi et al., 1978a; Noguchi et al., 1978b; Takata, 1981; Neu and Labthavikul, 1982). This report summarizes our in vitro evaluation of apalcillin. Apalcillin was compared to five other penicillins and two third-gen- eration cephalosporins against representative bacterial isolates collected from six separate medical centers. Additional tests were performed to evaluate [3-1actamase hydrolysis rates and [3-1actamase inhibition.

MATERIALS AND METHODS

Antimlcrobia l Agents

Apalcillin was provided as an assayed standard powder by Wyeth Laboratories (Phil- adelphia, PA). Other comparative antimicrobial agents were provided by the appro- priate pharmaceutical companies.

From The Clinical Microbiology Institute, (A.L.B.); Kaiser Permanente Regional Laboratory {Oregon Region), Clackamas, Oregon (R.N.J.}; and Centers for Disease Control, Atlanta, Georgia {C.T.)

Address reprint requests to: Arthur L. Barry, Ph.D., Director, The Clinical Microbiology Institute, 9595 S.W. Tualatin/Sherwood Hwy., P.O. Box 947, Tualatin, OR 97062.

Received February 20, 1984; revised and accepted May 4, 1984.

© 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue. New York, NY 10017 0732-8893/85/$O3.00

8 A.L. Barry et al.

Microorganisms

Representative samples of various common bacterial species, were collected from six geographically separate medical centers and additional isolates were contributed by the Centers for Disease Control, Atlanta, Georgia. The culture collection was obtained from The Cleveland Clinic Foundation, Cleveland, Ohio; Northwestern Memorial Hospital, Chicago, Ill.; University of California, Davis, Medical Center, Sacramento, Calif.; St. Francis Hospital, Wichita, Kans.; Kaiser-Permanente Regional Laboratory, Clackamas, Oregon; St. Vincent Hospital and Medical Center, Portland, Oregon.

Antimicrobial Susceptibility Tests

Broth microdilution tests were utilized for these comparative studies (National Com- mittee for Clinical Laboratory Standards, 1982). Eight different drugs were tested against each representative strain. Twofold dilutions of the drugs were prepared in cation-supplemented Mueller-Hinton broth alld then dispensed into microdilution panels. The test panels were held at -20°C or colder, until needed. Once allowed to thaw at room temperature, each tray was inoculated with approximately 5 x 105 CFU/ml in each well. The minimal inhibitory concentration (MIC) for each drug was recorded as the lowest concentration completely inhibiting visible bacterial growth after 16-18 hours at 35°C.

Fastidious' microorganisms were tested at the Centers for Disease Control, using the microdilution test panels, supplemented with 3% lysed horse blood (LHB) and NAD (25 ~g/ml) for Haemophilus inf/uenzae or with 3% LHB for testir~g Neisseria

• meningitidis, [~-hemolytic streptococci and Streptococcus pneumoniae. For testing Neisseria gonorrhoeae, an agar dilution technique was used with proteose peptone agar No. 3 supplemented with 1% haemoglobin and 1% Kellogg's supplement.

D-Lactamase Studies

~-lactamase hydrolysis rates were determined with crude extracts from strains known to produce different types of ~-lactamases (Richmond and Sykes, 1973), according to the methods described earlier (Barry et al., 1980; Barry and Jones, 1982). Hydrolysis rates were measured with a scanning UV spectrophotometer, using 230-235 nm at 37°C. Data were expressed as relative hydrolysis rates, compared to that of benzyl- penicillin (arbitrarily defined as 100%). Reaction mixtures were prepared to contain 1.0 ml of 10 -3 M ~-lactam substrate in 0.05 M pH 7.0 phosphate buffer.

[3-1actamase inhibition studies were performed by adding five different concen- trations of the test drug to the substrate (0.1-1000% of the test substrate). The ref- erence ~-lactamase-labile chromogenic cephalosporin was pyridine-2-azo-p-dime- thylanaline cephalosporin (PADAC) (Jones et al., 1982), tested at a concentration of 0.5 x 10 4 M (31.1 ~g/ml) in pH 7.0, 0.05 M phosphate buffer. Changes in the reaction mixtures at 37°C were followed by using a cer~trifugalfast analyzer (CentrifiChem 400, Union Carbide, Tarrytown, NY) monitored at 570 nm.

RESULTS

Comparative MICs with Representative Strains

Dilution tests were performed with representative clinical isolates. These tests were performed at The Clinical Microbiology Institute, Tualatin, Oregon and at the Centers for Disease Control, Atalanta, Georgia. Internal control data confirmed comparability

A p a l c i l l i n 9

T A B L E 1 . I n V i t r o A c t i v i t y o f A p a l c i l l i n a n d S e v e n O t h e r B - L a c t a m s A g a i n s t 1 5 S p e c i e s o f

C o m m o n G r a m - N e g a t i v e B a c i l l i

A n t i m i c r o b i a l MIC Rar lge

O r g a n i s m (no . t e s t e d ) a g e n t (~g /ml ) MICso MICgo

Escherichia coil (30) A p a l c i l l i n ~<0.5 - > 2 5 6 1 .0 > 2 5 6

A z l o c i l l i n 2 .0 - > 2 5 6 8 .0 > 2 5 6

C a r b e n i c i l l i n 1 .0 - > 2 5 6 8 .0 > 2 5 6

M e z l o c i l l i n 1.0 - ~ 2 5 6 2 .0 > 2 5 6

P i p e r a c i l l i n ~ 0 . 5 - > 2 5 6 2 ,0 > 2 5 6

T i c a r c i l l i n ~<0.5 - > 2 5 6 2 .0 > 2 5 6

C e f o t a x i m e ~ 0 . 0 6 - 1 .0 <~0,06 0 . 1 2

C e f o p e r a z o n e ~ 0 . 0 6 - 64 0 ,12 4 .0

Citrobacter diversus (20)

Citrobacter freundii (21)

Enterobacter a e r o g e n e s (20)

Enterobacter agglomerans (20)

Enterobacter cloacae (20)

A p a l c i l l i n ~<0.5 - 2 5 6 8 .0 32

A z l o c i l l i n 8 .0 - > 2 5 6 32 1 2 8

C a r b e n i c i l l i n 1.0 - > 2 5 6 1 2 8 > 2 5 6

M e z l o c i l l i n 2 .0 - 2 5 6 8 ,0 32

P i p e r a c i l t i n 1 .0 - 1 2 8 8 .0 32

T i c a r c i l l i n 1 .0 - > 2 5 6 1 2 8 > 2 5 6

C e f o t a x i m e ~ 0 . 0 6 - 0 .25 ~<0.06 0 .25

C e f o p e r a z o n e ~ 0 . 0 6 - 2 .0 ~<0.06 0 .5

A p a l c i l l i n ~ 0 . 5 - > 2 5 6 1 .0 6 4

A z l o c i l l i n 4 .0 - > 2 5 6 16 1 2 8

C a r b e n i c i l l i n 2 .0 - > 2 5 6 4 .0 > 2 5 6

M e z l o c i l l i n 1.0 - > 2 5 6 4 .0 6 4

P i p e r a c i l l i n 1 .0 - > 2 5 6 2 .0 6 4

T i c a r c i l l i n 1 .0 - > 2 5 6 2 .0 > 2 5 6

C e f o t a x i m e ~ 0 . 0 6 - 0 .5 0 .12 0 .5

C e f o p e r a z o n e 0 .12 - 1.0 0 .25 1 .0

A p a l c i l l i n 1.0 - 1 2 8 2 .0 6 4

A z l o c i l l i n 16 - > 2 5 6 32 > 2 5 6

C a r b e n i c i l l i n 2 .0 - > 2 5 6 8 .0 > 2 5 6

M e z l o c i l l i n 4 .0 - > 2 5 6 4 .0 1 2 8

P i p e r a c i l l i n 2 .0 - 1 2 8 4 .0 1 2 8

T i c a r c i l l i n 1.0 - > 2 5 6 2 .0 1 2 8

C e f o t a x i m e ~ 0 . 0 6 - 16 ~<0.06 8 .0

C e f o p e r a z o n e ~ 0 . 0 6 - 32 0 .25 8 .0

A p a l c i l l i n 0 .5 - 8 .0 1 .0 4 .0

A z l o c i l l i n 1 .0 - 64 8 .0 32

C a r b e n i c i l l i n 0 .5 - 8 .0 1.0 4 .0

M e z l o c i t l i n 0 .5 - 16 2 .0 8 .0

P i p e r a c i l l i n 0.5 - 16 1.0 4 . 0

T i c a r c i l l i n 0 .5 - > 2 5 6 4 .0 > 2 5 6

C e f o t a x i m e ~ 0 . 0 6 - 0 .5 <~0.06 0 .25

C e f o p e r a z o n e ~ 0 . 0 6 - 4 .0 0 .25 1 .0

A p a l c i l l i n 0 .5 - 64 2 .0 32

A z l o c i l l i n 8 .0 - > 2 5 6 16 < 2 5 6

C a r b e n i c i l l i n 1 .0 - 2 5 6 8 .0 64

M e z l o c i l l i n 1.0 - 32 4 .0 16

P i p e r a c i l l i n 1 .0 - 32 2 .0 32

T i c a r c i l l i n 1.0 - 2 5 6 2 .0 6 4

C e f o t a x i m e ~ 0 . 0 6 - 9 6 4 0 .25 32

C e f o p e r a z o n e ~ 0 . 0 6 - 9 6 4 0 .5 32

( c o n t i n u e d )

10 A.L. B a r r y e t al .

T A B L E 1. I n V i t r o A c t i v i t y of A p a l c i l l i n a n d S e v e n O t h e r ~ - L a c t a m s A g a i n s t 15 S p e c i e s of

C o m m o n G r a m - N e g a t i v e B a c i l l i (continued)

Ant imic rob i a l MIC Range

Organ i sm (no. tes ted) agent (p,g/ml) MICso MIC9o

KJebsiella spp.

K. pneumoniae (30)

K. oxytoca (10)

Serratia m a r c e s c e n s (30)

Pro teus mirabilis (30)

Pro teus vulgaris (20)

Morganel|a morganii (19)

Providencia re t tger i (20)

A p a l c i l l i n

A z l o c i l l i n

Ca rben i c i l l i n

M e z l o c i l l i n

P ipe r a c i l l i n

T ica rc i l l i n

Cefo tax lme

Cefoperazone

A p a l c i l l i n

A z l o c i l l i n

Ca rben ic i l l i n

Me z loc i l l i n

P ipe rac i l l i n

T ica rc i l l i n

Cefo tax lme

Cefoperazone

A p a l c i l l i n

A z l o c i l l i n

Ca rben ic i l l i n

Me z loc i l l i n P ipe rac i l l i n

T ica rc i l l i n

Cefo tax lme

Cefoperazone

A p a l c i l l i n

A z l o c i l l i n

C a r b e n i c i l l i n

M e z l o c i l l i n

P i p e r a c i l l i n

T i c a r c i l l i n

Cefo tax lme

Cefoperazone

A p a l c i l l i n A z l o c i l l i n

C a r b e n i c i l l i n M e z l o c i l l i n

P i p e r a c i l l i n

T i c a r c i l l i n

Cefo tax lme Cefoperazone

A p a l c i l l i n A z l o c i l l i n

C a r b e n i c i l l i n M e z l o c i l l i n

P i p e r a c i l l i n T i c a r c i l l i n Cefo tax ime Cefoperazone

2.0 - > 2 5 6 8.0 64

16 - > 2 5 6 32 256

32 - > 2 5 6 256 > 2 5 6

4.0 - > 2 5 6 8.0 256

4.0 - > 2 5 6 8.0 64

32 - > 2 5 6 128 > 2 5 6

~<0.06 - 0.25 ~<0.06 ~<0.06

0.12 - 64 0.25 2.0

2.0 - 64 4.0 16

8.0 - 256 16 128

2.0 - 128 8.0 64

1.0 - 64 4.0 16

0.5 - 32 2.0 16

2.0 - > 2 5 6 8.0 32

0.12 - 4 . 0 0.25 2.0

0.5 - 32 1.0 8.0

~<0.5 - 1.0 ~<0.5 ~<0.5 1.0 - 4 . 0 2.0 2.0

~<0.5 - 2.0 ~<0.5 ~<0.5

~<0.5 - 1.0 ~<0.5 ~<0.5 ~<0.5 - ~<0.5 ~<0.5 ~<0.5

~<0.5 - 1.0 ~<0.5 ~<0.5

~ 0 . 0 6 - ~<0.06 ~<0.06 ~<0.06 0.25 - 0 . 5 0.5 0.5

0.5 - > 2 5 6 2.0 8.0 2.0 - > 2 5 6 8.0 32

1.0 - 64 4.0 64

0.5 - > 2 5 6 1.0 8.0 0.5 - 8.0 1.0 2.0

0.5 - 64 4.0 64

~<0.06 - 0.5 ~<0.06 0.12

0.25 - 8.0 1.0 2.0

0.5 - > 2 5 6 8.0 256

2.0 - > 2 5 6 8.0 > 2 5 6

0.5 - 256 1.0 8.0

0.5 - > 2 5 6 2.0 64

0.5 - > 2 5 6 1.0 32

0.5 - 256 4.0 16

~<0.06 - 8.0 0.12 4.0 0.25 - 32 1.0 16

0.5 - > 2 5 6 2.0 > 2 5 6 2,0 - > 2 5 6 8.0 > 2 5 6 0.5 - > 2 5 6 1.0 > 2 5 6 1.0 - > 2 5 6 2.0 256

0.5 - > 2 5 6 1.0 128 0.5 - > 2 5 6 1.0 > 2 5 6

~<0.06 - 0.25 ~ 0 . 0 6 0 .25

~<0.06 - 64 0.5 64

Apalcillin 11

TABLE 1. In Vitro Activity of Apalcillin and Seven Other ~-Lactams Against 15 Species of Common Gram-Negative Bacilli (continued)

A n t i m i c r o b i a l MIC R a n g e

O r g a n i s m (no. t e s t ed ) a g e n t (~g /ml ) MICso MICgo

Providencia stuartii (20) A p a l c i l l i n 0.5 - > 2 5 6 2.0 256

A z l o c i l l i n 2.0 - > 2 5 6 32 > 2 5 6

C a r b e n i c i l l i n 0.5 - > 2 5 6 2.0 > 2 5 6

M e z l o c i l l i n 1.0 - > 2 5 6 4.0 256

P i p e r a c i l l i n 0.5 - > 2 5 6 2.0 128

T i c a r c i l l i n 0.5 - > 2 5 6 1.0 > 2 5 6

C e f o t a x i m e ~ 0 . 0 6 - 2.0 0.12 1.0

C e f o p e r a z o n e 0 .12 - 32 1.0 16

Acinetobacter calcoaceticus ssp . antitratus (15)

ssp. lwoffii (5)

Pseudomonas aeruginosa (25)

A p a l c i l l i n 1.0 - 16 2.0 4 .0

A z l o c i l l i n 8.0 - 128 32 64

C a r b e n i c i l l i n 2.0 - 32 4.0 8.0

M e z l o c i l l i n 8.0 - 64 32 32

P i p e r a c i l l i n 4.0 - 16 8.0 16

T i c a r c i l l i n 1.0 - 4 .0 4.0 4 .0

C e f o t a x i m e 2.0 - 16 8.0 16

C e f o p e r a z o n e 8.0 - 64 32 64

A p a l c i l l i n ~ 0 . 5 - 4 .0 1.0 4.0

A z l o c i l l i n 2.0 - 32 4.0 16

C a r b e n i c i l l i n 8.0 - 128 16 32

M e z l o c i l l i n 4.0 - 64 8.0 32

P i p e r a c i l l i n 1.0 - 8.0 2.0 8.0

T i c a r c i l l i n 4.0 - 64 8.0 16

C e f o t a x i m e 2.0 - 32 8.0 32

C e f o p e r a z o n e 2.0 - 16 4.0 16

between broth microdilution tests performed in the two laboratories and, thus, data from the two laboratories were combined before analysis.,

Table 1 summarizes the results of tests with 355 gram-negative bacilli comparing apalcillin to five penicillins, cefotaxime, and cefoperazone. Proteus mirabilis strains were all susceptible to all study drugs but the other species included some strains that were resistant to the penicillins, i.e., maximal MICs/> 64 ~g/ml. Apalcillin MIC 50s were ~ 8.0 ~g/ml for all species groups, but MIC 90s were /> 32 ~g/ml with Escherichia coli, Citrobacter spp., Enterobacter spp., Klebsiella spp. Morganella spp., Providencia rettgeri, and P. stuartii. Piperacillin, mezlocillin, and apalcillin dem- onstrated similar activity against the Enterobacteriaciae.

Against Acinetobacter spp., apalcillin was approximately twice as active as ti- carcillin or carbenicillin. The other ]3-1actams, including piperacillin, were signifi- cantly less effective. Against P. aeruginosa, apalcillin was twice as active as piper- acillin, with MIC 90s of 4.0 and 8.0 ~g/ml~ respectively.

Comparative data with Staphylococcus aureus and coagulase negative Staphy- lococcus species are summarized in Table 2. As expected, methicillin-resistant strains of staphylococci were relatively resistant to all of the 13-1actams, as were other f3- lactamase-positive staphylococci. Although the methicillin-resistant S. aureus strains appeared to be susceptible to ' ~ 16 p,g/ml, they should be considered resistant to the penicillins and cephalosporins. With all six penicillins that were studied, penicillin-

12 A.L. Barry et al.

TABLE 2. In Vitro Ac t iv i ty of Six Pen ic i l l ins and T w o Cepha lospor ins Agains t [3-Lactamase-Posit ive and -Negat ive Staphylococcus spp. and Haemophilus influenzae

MIC (p.g/ml) for 50% and 90% of strains within each subgroup °

S. aureus S. epidermidis H. influenzae

Antimicrobial Pen S Pen R Meth R Pen S Pen R Meth R Amp S Amp R agents (23) b (27) (10) (9) (10) (5) (30) (31)

MICs0 for: Apalcillin ~< 0.5 8.0 8.0 ~< 0.5 2.0 64 ~ 0.5 4.0 Azlocillin ~< 0.5 16 8.0 ~< 0.5 2.0 64 ~ 0.5 8.0 Carbenicillin ~ 0.5 4.0 16 1.0 2.0 >256 ~ 0.5 2.0 Mezlocillin ~< 0.5 8.0 8.0 ~< 0.5 2.0 64 ~ 0.5 4.0 Piperacillin ~< 0.5 8.0 16 ~< 0.5 2.0 128 ~ 0.5 2.0 Ticarcillin ~< 0.5 4.0 8.0 1.0 2.0 >256 ~ 0.5 2.0 Cefotaxime 1.0 2.0 8.0 1.0 0.5 >32 ~ 0.06 ~< 0.06 Cefoperazone 1.0 2.0 8.0 1.0 1.0 >32 ~ 0.06 ~< 0.06

MICg0 for: Apalcillin 1.0 128 16 1.0 8.0 256 ~ 0.5 64 Azlocillin 2.0 128 8.0 1.0 8.0 128 ~ 0.5 128 Carbenicillin 2.0 8.0 16 4.0 32 >256 ~ 0.5 8.0 Mezlocillin 2.0 128 8.0 1.0 8.0 >256 ~ 0.5 64 Piperacillin 2.0 128 16 1.0 8.0 256 ~ 0.5 32 Ticarcillin 2.0 8.0 16 4.0 32 >256 ~< 0.5 16 Cefotaxime 2.0 2.0 16 4.0 4.0 >32 ~ 0.06 ~ 0.06 Cefoperazone 2.0 4.0 16 4.0 2.0 >32 ~ 0.06 ~< 0.06

°Abbreviations: Resistant (R) or susceptible (S) to methicillin (Meth), penicillin (Pen) or ampicillin (Amp). bNumber of strains within each group is noted in parentheses.

res i s tan t s t a p h y l o c o c c i p r o v i d e d MICs that were m u c h greater than those ob ta ined w i t h p e n i c i l l i n - s u s c e p t i b l e strains. Because al l of the tes ted pen i c i l l i n s are inact i - va t ed by s t a p h y l o c o c c a l pen ic i l l i nase , pen ic i l l in - res i s t an t s trains shou ld be cons id - e red res is tan t e v e n t h o u g h the MICs migh t be w i t h i n the range of ach i evab le b lood levels . T h e cepha lo spo r in s , on the o ther hand, are res is tant to s t aphy lococca l [3- lac tamases ; t hey w e r e a lmos t equa l ly ac t ive against pen ic i l l in - res i s t an t and pen ic i l -

l i n - suscep t ib l e strains. A m p i c i l l i n - s u s c e p t i b l e H. influenzae were ve ry suscep t ib le to all of the [3-1actams

s t ud i ed (MIC ~< 0.5 ~g/ml). The c e p h a l o s p o r i n s were also ac t ive against ampic i l l i n - res i s tan t H. influenzae, but the pen i c i l l i n s were re la t ive ly inef fec t ive against these [3-1actamase-producing s t ra ins (Table 2). Ten strains of H. influenzae were fur ther tes ted w i t h three i n o c u l u m dens i t i e s (Table 3). Wi th an i n o c u l u m of 103 or 105 CFU/ml , B- lac tamase-pos i t ive s trains appea red to be suscep t ib le to ach ievab le con- cen t ra t ions of each drug. H o w e v e r , w h e n the i n o c u l u m was inc reased to 107 CFU/ ml, [3-1actamase-posit ive s t ra ins w e r e very res is tant and B- lac tamase-negat ive s trains r e m a i n e d suscep t ib l e to all of the pen ic i l l ins .

[3-1actamase-negative and -pos i t ive s trains Of N. gonorrhoeae were tes ted in an agar d i l u t i o n s y s t e m (Table 4). Apa lc i l l i n , ampic i l l i n , t icarc i l l in , and e r y t h r o m y c i n w e r e tes ted agains t 61 isolates. Al l four drugs were very ac t ive against [3-1actamase- nega t ive strains. B- lac tamase-pos i t ive s trains were re la t ive ly res is tant to all th ree p e n i c i l l i n s bu t w e r e suscep t ib l e to e ry th romyc in . In the s u p p l e m e n t e d m e d i u m used for these s tud ies (1% h e m o g l o b i n and 1% Kel logg 's supp lement ) , five cont ro l s trains p r o v i d e d apa l c i l l i n MICs two- to four- fo ld h igher than those ob ta ined in n n s u p p l e -

Apalci l l in 13

TABLE 3. Effect of Increasing Inoculum Concentration on the In Vitro Activity of Seven Penici l l ins Against Five ~-Lactamase-Negative a and Five ~-Lactamase- Positive Haemophilus injf]uenzae

Antimicrobial MICs0 with inoculum b of:

agent 13-1actamase 103 l0 s 107

Ampicillin Neg ° 0.25 0.25 0.25 Pos 1.0 2.0 >32

Apalcillin Neg ~<0.25 ~<0.25 ~<0.25 Pos 0.5 1.0 >128

Piperacillin Neg ~<0.25 ~<0.25 ~<0.25 Pos ~<0.25 0.5 >128

Mezlocillin Neg ~<0.25 ~<0.25 ~<0.25 Pos 1.0 2.0 >128

Azlocillin Neg ~<0.25 ~<0.25 ~<0.25 Pos 0.5 2.0 >128

Carbenicillin Neg ~<0.25 ~<0.25 ~<0.25 Pos ~<0.25 0.5 >128

Ticarcillin Neg ~<0.25 <~0.25 ~<0.25 Pos ~<0.25 0.5 128

°Includes two 13-1actamase-negative strains with elevated ampillin MICs (2---4 ~g/ml); they were also relatively resistant to carbenicillin and ticarcillin (1-4 pg/ml), but susceptible to the other penicillins {MIC ~<0.25 ~g/ml).

blnoculum expressed as colony forming units per milliliter (CFU/ml).

mented Mueller-Hinton agar. Supplementat ion with 1% Isovitalex and 1% hemoglo- b in also yielded discrepant results compared to unsupplemented media. Unsupple- mented proteose peptone agar yielded MICs that were the same as those in unsupp lemented Mueller-Hinton agar.

Table 5 summarizes in vitro susceptibility data with representative N. meningit idis and four different Streptococcus species. Included were 13 strains of S. pneumoniae that were relatively resistant to benzyl-penici l l in (MIC > 0.06 ~g/ml}; they appeared to be susceptible to mezloci l l in but relatively resistant to the other penicil l ins. Apal-

TABLE 4. Susceptibil i ty of 13-Lactamase-Producing and Nonproducing N. gonorrhoeoe to Four Antimicrobial Agents

MIC (p.g/ml} for"

Antimicrobial agent 13-1actamase 50% 90%

Apalcillin Negative ~<0.5 ~<0.5 Positive 1.0 2.0

Ampicillin Negative ~<0.12 0.5 Positive 8.0 >32

Ticarcillin Negative ~<0.5 1.0 Positive 8.0 >32

Erythromycin Negative 0.25 1.0 Positive 0.25 1.0

°Percent of strains, based on tests with 30 ~-lactamase-positive and 31 ~-lactamase-negative strains of N. gonorrhoeae.

14 A.L. Barry et al.

TABLE 5. In Vitro Activity of Six Penicillins and Two Cephalosporins Against Four Streptococcus species and N. meningitidis.

N. meningitidis S. agalactiae S. pyogenes S. p n e u m o n i a e S. faecalis Antimicrobial agents (25) (22) (21) (26) b (25)

Apalcil l in ~<0.5 ~<0.5 ~<0.5 1.0 1.0 Azlocillin ~<0.5 ~<0.5 ~<0.5 1.0 1.0 Carbenicillin ~<0.5 2.0 1.0 16 32 Mezlocillin <~0.5 ~<0.5 ~<0.5 ~<0.5 <0.5 Piperacill in 40.5 ~<0.5 ~<0.5 1.0 2.0 Ticarcillin <~0.5 2.0 ~<0.5 16 32 Cefotaxime ~<0.06 ~<0.06 0.12 0.25 >64 Cefoperazone 0.12 0.25 0.25 1.0 32

aNumber of strains noted in parentheses. blncluding 13 strains relatively resistant to benzylpenicillin [MIC > 0.06 jzg./ml).

cillin inhibited S. faecalis (MIC 90 1.0 Ixg/ml), as did azlocillin, mezlocillin, and piperacillin. Carbenicillin, ticarcillin, cefotaxime, and cefoperazone were not active against S. faecalis (MIC 90/> 32 ~.g/ml).

13-Lactamase Hydrolysis/Inhibition 13-1actamase hydrolysis rates for five different penicillins are compared in Table 6. Seven different enzyme preparations were tested, using benzylpenicillin as the stan- dard reference drug. As expected, the 13-1actamases that are primarily cephalospor- inases were relatively ineffective against the penicillins. Apalcillin, piperacillin, and azlocillin were similar in their susceptibility to hydrolysis by most 13-1actamases. Ticarcillin was most slowly hydrolyzed. Apalcillin and ticarcillin were relatively resistent to the PSE 2 enzyme preparation.

The ability of the penicillins to inhibit the hydrolytic activity of Type I (P 99) 13- lactamase produced by E. cloacae is described in Table 7. Moderate inhibition of

TABLE 6. 13-Lactamase Hydrolysis Rates of Apalcillin Compared to Those of Five Other Penicillins

Ampici l l in RHR b compared to benzyl penici l l in Organism and

13-1actamase type p,M/min ° RHR b Apal c Piper Azlo Ticar Diclox

Bacillus cereus Pen'ase c 7512 457 77.1 132 114 17.8 <1.0 Enterobacter cloacae, P99 14.8 88.6 25.7 28.1 17.1 3.6 8.4 Escherichia coli, TEM-1 623 299 54.8 63.9 65.9 11.2 <1.0 Escherichia coli, TEM-2 1062 410 80.7 66.8 81.5 8.6 <1.0 Escherichia coil, OXA-2 26.6 182 142 80.1 125 28.8 14.7 Klebsiella oxytoca, K1 548 182 77.0 24.6 51.3 8.1 1.6 Pseudomonas aeruginosa, PSE-2 7.8 96.8 5.3 135 108 3.7 12.4

aThe ~,M/min/ml of partially purified 13-1actamase preparation, hydrolysis rates for ampicillin are presented to define the potency of each enzyme preparatin.

bRHR = relative hydrolysis rate compared to that of benzyl penicillin, expressed as a value of 100%. ~Abbreviations: Apal = apalcillin; Piper = piperacil l in; Azlo = azlocillin; Ticar = ticarcillin; Di-

clox = dicloxacillin. CPen'ase = commercial penicillinase from B. cereus (BBL, Microbiology systems, Cockeysville, MD).

Apalci l l in 15

TABLE 7, Inhibi t ion of ~-Lactamase Hydrolysis of PADAC by Equimolar Concentrations of Apalci l l in and Comparison ~-Lactams

PADAC RHR combined with: ° Organism and

~-lactamase type Apal Piper Ticar Diclox Cefot

Enterobacter cloacae, P99 <1.0 <1.0 <1.0 <1.0 <1.0 Pseudomonas aeruginosa, PSE-2 30 57 54 36 60 Escherichia cell, TEM-I 35 55 35 22 100

Escherichia cell, TEM-2 40 57 26 35 100 K|ebsJella oxytoca, K1 54 30 76 85 95 Escherichia coli, OXA-2 70 55 58 100 75 Bacillus cereus Pen'ase b 85 80 100 100 100

Abbreviations: Apal = apalcillin, Piper = piperacillin; Ticar = ticarcillin; Diclox = cicloxacillin; Ce- fot = cefotaxime.

°Relative hydrolysis rate (RHR) of pyridine-2.azo-p-dimethylanaline cephalosporin {PADAC) combined with the indicated 13-1actam, compared to that of PADAC alone, with no additional 13-1actam {100%}.

bPen'ase = commercial penicillinase from B. cereus {BBL Microbiology Systems, Cockeysville, MD}.

1011

:E

75

t r

.w

-~ 50 > , -1-

:> ",,=

~ 25 PSE2

K1

Np99 TEM2 o:1 11o lo 1do lobe

Inhibitor Concentration as % of PADAC Conc.

Figure 1. Pattern of ~-lactamase inhibition by apalcillin using seven bacterial enzymes and 0.5 × 10 -4 M PADAC as the enzyme-labile substrate. Refer to Tables 6 and 7 for enzyme clas- sifications and bacterial strains.

16 A.L. Barry et al.

five other enzymes obtained from gram-negative bacilli was also observed with apal- cillin, piperacillin, and ticarcillin. Figure 1 displays inhibition of seven different B- lactamases by different concentrations of apalcillin. P 99 is inhibited by relatively low concentrations of apalcillin but significant inhibition (~< 50%) of the other en- zymes requires fairly large amounts of apalcillin.

DISCUSSION

Apalcillin has a spectrum of antibacterial activity that is very similar to that of piperacillin, except that apalcillin is significantly more active against Pseudomonas aeruginosa and Acinetobacter spp. In the current study, the few bacterial isolates that were resistant to piperacillin were also resistant to apalcillin. Against the en- terobacteriaceae, mezlocillin had a spectrum of activity similar to that of piperacillin and apalcillin. This study confirms our previous observation (Barry et al., 1980) that azlocillin and piperacillin have similar activity against Pseudomonas spp.: apalcillin is significantly more active. Against Acinetobacter spp., apalcillin is uniquely active, compared to the other penicillins and comparison drugs.

Like the other penicillins, apalcillin is susceptible to hydrolysis by the common ~-lactamase enzymes (Noguchi et al., 1978a; Noguchi et al., 1978b; Takata et al., 1981; Barry and Jones, 1982; Neu and Labthavikul, 1982). Noguchi and colleagues (1978a, 1978b) have shown that the increased activity of apalcillin is due to its high affinity for penicillin-binding proteins and its ability to inhibit peptidoglycan syn- thesis. Because of its mode of action, apalcillin should be expected to be bactericidal at concentrations equal to or slightly greater than the MIC. Strains with low levels of constituative ~-lactamase may be inhibited and killed before the drug is signifi- cantly hydrolyzed by the enzyme (Takata et al., 1981). Some of the common plasmid- mediated ~-lactamase-producing strains appear to be susceptible to apalcillin in vitro. However, they should produce marked discrepancies between MIC and MBC values and should show a marked inoculum effect (Noguchi et al., 1978a; Neu and Lab- thavikol, 1982). Such in vitro characteristics would not be expected to occur with ~-lactamase negative isolates. The ability of apalcillin to inhibit hydrolytic activity of various [3-1actamases is, to some extent, related to the affinity of the drug for the enzymes. The practical significance of this [3-1actamase inhibition remains unclear.

Because of its mode of action and mechanisms of resistance, one might expect a potentiation of activity of apalcillin when it is used in combination with other an- timicrobial agents. Neu and Labthavikul (1982) reported synergy or partial synergy when apalcillin was combined with tobramycin or amikacin. In their study, strains that were resistant to apalcillin or to the aminoglycoside were rendered susceptible to the combination. Further study with various drug combinations would seem ap- propriate, especially with the goals of potentiating the activity of apalcillin against [3-1actamase-producing strains and of developing a bactericidal activity against S. faecalis.

Blood levels achieved with apalcillin compare favorably to those with piperacillin (Lode et al., 1984). However, apalcillin has a prolonged serum half-life and it is primarily excreted by nonrenal routes. Urine levels of piperacillin are substantially greater than those seen with apalcillin.

The broad spectrum of antibacterial activity demonstrated by apalcillin suggests that it may be a promising therapeutic agent. Its increased activity against P. aeru- ginosa and Acinetobacter spp. may represent a significant advantage over pipera- cillin. Its relative instability to some [3-1actamases represents a major disadvantage as compared to cefotaxime and other third-generation cephalosporins. On the other

Apalc i l l in 17

hand, the cepha lospor ins are less active against enterococci, Acinetobacter spp., and Pseudomonas spp.

REFERENCES

1. Barry AL, Jones RN {1982) A three category system for interpretation of disk tests for Pseudomonas-active penicillins and ~-lactamase hydrolysis/inhibition studies. ] Antimi- crob Chemother 9 {Suppl. A}:35.

2. Barry AL, Thornsberry C, Jones RN, Gavan TL {1980) In vitro activity of mezlocillin and azlocillin compared with that of four other penicillins and two aminoglycosides. C/eveland Clin Quart 47:311.

3. Bodey GP, Weaver S, Pan T (1978} PC-904, a new semisynthetic penicillin. Antimicrob Agents Chemother 13:14.

4. Jones RN, Wilson HW, Novick WJ (1982} In vitro evaluation of pyridine-2-azo-p-dimethy- laniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with ni- trocefin, cephacetrile, and other beta-lactam compounds. ] Clin Microbiol 15:677.

5. Lode H, Elvers A, Koeppe P, Borner K {1984} Comparative pharmacokinetics of apalcillin and piperacillin. Antimicrob Agents Chemother 25:105.

6. National Committee for Clinical Laboratory Standards (1982} Tentative Standard M7-T. Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aer- obically. National Committee for Clinical Laboratory Standards, Villanova, Pennsylvania.

7. Neu HC, Labthavikul P {1982} In vitro activity of apalcillin compared with that of other new penicillins and anti-Pseudomonas cephalosporins. Antimicrob Agents Chemother 21:906.

8. Noguchi H, Eda Y, Tobiki M, Nakagome T, Komatsu T (1976} PC-904, a novel broad- spectrum semisynthetic penicillin with marked antipseudomonal activity: Microbiological evaluation. Antimicrob Agents Chemother 9:262.

9. Noguchi H, Kubo M, Kurashige S, Mitsuhashi S (1978a} Antibacterial activity of apalcillin (PC-904} against gram-negative bacilli, especially ampicillin-, carbenicillin-, and genta- micin-resistant clinical isolates. Antimicrob Agents Chemother 13:745.

10. Noguchi H, Matsuhashi M, Takaoka M, Mitsuhashi S (1978b) New antipseudomonal pen- icillin, Pc-g04: Affinity to penicillin-binding proteins and inhibition of the enzyme cross linking peptidoglycan. Antimicrob Agents Chemother 14:617.

11. Richmond MH, Sykes RB (1973} The beta-lactamases of gram negative bacteria and their possible physiologic role. Adv Microb Physiol 9:31.

12. Takata N, Suginaka H, Kotani S, Ogawa M, Kosaki G {1981}. 13-1actam resistance in Serratia marcescens: Comparison of action of benzylpenicillin, apalcillin, cefazolin and ceftizoxime. Antimicrob Agents Chemother 19:397.