425
DIAGN MICROBIOL INFECT DIS 1991;14:425-434
In vitro Activity Evaluations of Cefdinir (FK482, CI-983, and PD134393) A N o v e l Orally A d m i n i s t e r e d Cephalosporin Beth M. Briggs, Ronald N. Jones, Meridith E. Erwin, Mary S. Barrett, and David M. Johnson
Cefdinir, a so-called third-generation oral cephalosporin was tested in vitro against over 700 pathogens from patients with bacteremia. Cefdinir was very active against the Enterobacteriaceae with a 50% minimum inhibitory concentration (MICso) value range of 9 replicates) were (a) Escherichia coli ATCC 25922 = 0.25 ~g/ml, (b) Enterococcus faecalis ATCC 29212 = 1 ~g/ml, (c) S. aureus ATCC 29213 = 0.25 ~g/ml, and (d) P. aeruginosa ATCC 27853 > 16 ~g/ml (NCCLS, 1990b).
Antimicrobial Susceptibility Testing Methods The NCCLS (1987, 1989, and 1990a and b) technical recommendations were employed for all aspects of the study. The MICs were determined mainly through the use of the broth microdilution method (NCCLS,
1990b), although the agar dilution was used to enable testing of Neisseria spp. (Jones et al., 1989) and some intermethod experiments, as for example, broth versus agar. Mueller-Hinton medium was used for the majority of tests, whereas GC agar base (supplemented with an XV-type reagent) (NCCLS, 1990b) was used for the N. gonorrhoeae isolates. Haemophilus test medium (HTM) (Jorgensen et al., 1987 and 1990) was the medium of choice for fastidious organism susceptibility testing including H. influenzae, pneumococci, and other streptococci. The Wilkins-Chalgren agar medium was used for the anaerobic bacteria testing with the NCCLS (1989) agar dilution method at an inoculum of 105 colony-forming units (CFU)/spot. Using end-point rejection standards documented by Pearson et al. (1980), minimum bactericidal concentration (MBC) results were gathered utilizing NCCLS (1987), methods from subculturing broth microdilution trays. To study inoculum concentration effects on cefdinir, MICs employed concentrations of ]03-106 CFU/spot. The incubation environment, supplements, and medium pH were also modified (Table 1).
Other Procedures Permeability of the outer membrane of E. coli strains DCO and its mutant DC2 were tested using the method of Richmond and colleagues (Richmond et al., 1976; Richmond and Wooton, 1976). The test strains were provided by Dr. Seibert of Hoechst AG (FRG).
RESULTS Antimicrobial Activity Against GramNegative Bacteria Table 2 contains a summary of the cefdinir MIC results of various strains compared with those of four other cephalosporins. Fastidious species including N. gonorrhoeae (MICg0, ~ 16 p,g/ml) and Xanthomonas maltophilia (MICgo > 16 ~g/ ml). The anaerobic organisms of the B. fragilis gr. were also resistant to cefdinir (MICso, 16 ~g/ml).
C e f d i n i r in v i t r o A c t i v i t y
TABLE 1
427
Effect o f I n o c u l u m C o n c e n t r a t i o n s , S u p p l e m e n t s , a n d T y p e of I n c u b a t i o n o n t h e M I C s of C e f d i n i r T e s t e d A g a i n s t 29 S e l e c t e d B a c t e r i a Inoc. Conc. (CFU/Spot) 5% SBC b
Incub. 5% CO2
Mg 2÷c
16
8
4
4
8
>16
16
>16
16
>16
>16
>16
>16
>16
>16
Hafnia alvei, type I
4
16
>16
>16
8
16
Morganella morganii, type I
0.5
4
>16
4
4
4
Providencia stuartii, type I
1
1
>16
2
4
2
Serratia liquefaciens, type I
0.5
0.5
0.5
2
0.5
103
104
Acinetobacter anitratus
2a
2
Enterobacter aerogenes, type I
4
Organism/Mechanism a
cloacae, type Ia (P99)
marcescens, type I
16
>16
106
2 >16
>16
>16
>16
Escherichia coli TEM-1 TEM-2 HMS-1 OXA-1 OXA-2 OXA-3 SHV-1 ATCC 25922
0.12 0.06 0.12 0.25 0.12 0.12 O.06 0.25
0.12 0.12 0.25 0.25 0.25 0.12 0.12 0.25
2 2
2 2
0.06 2 1
0.12 0.12 0.25 0.5 0.25 0.25 0.12 0.25
0.25 0.06 0.25 0.25 0.25 0.12 0.12 0.25
0.25 0.25 0.25 0.5 0.5 0.25 0.25 0.25
0.12 0.06 0.25 0.25 0.25 0.12 0.12 0.25
>16 >16
2 2
4 4
2 2
0.12 4
0.12 >16
0.12 8
0.12 8
0.12 4
1
>16
1
2
1
>16 >16 >16 >16 >16
>16 >16 >16 >16 >16
>16 >16 >16 >16 >16
>16 >16 >16 >16 >16
>16 >16 >16 >16 >16
>16 >16 >16 >16 >16
>16
>16
>16
>16
>16
>16
Klebsiella oxytoca Type IVc (K1) Type IVc (K14)
Klebsiella pneumoniae Wild type ExSpBL e ExSpBL e
Pseudomonas aeruginosa CARB-1 CARB-2 CARB-4 OXA-4 ATCC 27853
Pseudomonas fluorescens Staphylococcus aureus Penase ATCC 29213
0.25 0.25
0.5 0.5
0.5 0.5
0.5 0.5
0.12 0.25
0.5 0.5
SBC, sheep blood ceils; ExSpBL, extended-spectrum [3-1actamase; Inoc. Conc., inoculum concentrations; and incub., incubation. aResistance mechanism ([3-1actamase type), if known. t'Inoculum at 104 CFU/spot. CMga+ content 10 x greater than normal agar, inoculum at 104 CFU/spot. aMIC in pzg/ml. eCapable of hydrolyzing cefotaxime and ceftazidime (Bush and Singer, 1989).
428
B.M. B r i g g s et al.
TABLE 2
Cefdinir Antimicrobial Activity Compared with Four Other Cephalosporins Tested Against Gram-Negative Bacteria MIC (~tg/ml)
Organism
No. Tested
Antimicrobial agent
50%
90%
Range
1 0.12 32 1 ~32 0.5-8 ~32 >32 > 32
0.12->16 0.12->4 1->32 0.5->32 ~ 32
Enterobacter aerogenes
20
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
0.5 0.5 8 16 ~ 16 >4 >32 >32 16
0.25-> 16 0.12->4 2->32 1->32 ~ 32
agglomerans
13
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
0.25 0.25 4 1 ~32 16 0.06->4 16-32 >32 ~0.25-1
Proteus mirabilis
19
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
~0.03 ~0.008 1 1 ~32
0.06-1 ~ 16 0.12->4 2->32 4->32 0.25->32
C e f d i n i r in vitro A c t i v i t y
TABLE 2
429
Continued MIC (p~g/ml) No.
Organism
Tested
Antimicrobial agent Cefuroxime Cefaclor Ceftriaxone
50%
90%
Range
>32 >32 ~0.25
>32 >32 ~0.25
>32 16->32 ~0.25
Providencia rettgeri
10
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
~0.03 ~0.008 0.25 16 ~0.25
0.5 0.25 8 >32 ~0.25
~32 ~4 >32 >32 2
~16 ~4 0.5->32 4->32 ~32 >32 2
4 >4 >32 >32 16
0.6-0.5 0.03-0.25 2-8 0.5-1 ~0.25 1->16 0.12->4 32->32 >32 ~0.25-16
0.12->16 2->4 0.25->32 8->32 ~0.25->32
Haemophilus influenzae
430
B.M. Briggs et al.
TABLE 2 C o n t i n u e d MIC (~g/ml) No.
Organism Moraxella catarrhalis ~-Lactamase positive
Tested
Antimicrobial agent
50%
90%
Range
33d
Cefdinir Cefixime
0.06 0.12
0.12 0.012
~0.015-0.12 ~0.015-0.12
12
Cefdinir Cefixime
0.03 0.03
0.06 0.12
~0.015-0.06 ~0.015-0.12
Neisseria gonorrhoeae ~-Lactamase positive ~-Lactamase negative
14 20e
Cefdinir Ce fdinir
~0.03 ~ 0.03
~0.03 ~ 0.03
~0.03 ~ 0.03-0.06
Neisseria spp.
16f
Cefdinir Penicillin
0.06 0.5
0.25 2
~ 0.015-0.25 0.064
Pseudomonas aeruginosa
30
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
> 16 >4 >32 >32 16
> 16 >4 >32 >32 > 32
> 16 >4 >32 >32 >32
> 16 >4 >32 >32 >32
0.12-> 16 4->4 2->32 G0.12->32 8->32
16
>16
~0.05->16
13-Lactamase negative
Xanthomonas maltophilia
10
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
Bacteroides fragilis gr.
37g
Cefdinir
> 16 >4 >32 >32 2-> 32
qncludes K. oxytoca (11 strains) and K pneumoniae (20 strains). blncludes S. flexneri (4 strains) and S. sonnei (6 strains). ~Ampicillin resistant by HTM MIC (~2 ~g/ml) or disk diffusion test (421 ram). qncludes BRO-1 (19 strains) and BRO-2 (14 strains) enzymes. qncludes 10 isolates of CMRNG and 10 isolates susceptible to penicillin (NCCLS, 1990a and b). fIncludes N meningitidis (5 strains), N subflava (6 strains), N. sicca (3 strains), and N mucosa (2 strains). qncludes B. fragilis (26 strains), B. distasonis (1 strains), B. ovatus (1 strain), B. vulgatus (3 strains), and B. thetaiotaomicron (6 strains).
The family Enterobacteriaceae species varied greatly in their susceptibility to cefdinir as well as to the other cephalosporins tested. Of the following enterics, ~90% had ~ 2 ~,g/ml cefdinir MIC values: Citrobacter diversus, Enterobacter agglomerans, E. coli, Klebsiella spp., Proteus mirabilis, Providencia rettgeri, Salmonella enteritidis, Shigella spp., and Yersinia enterocolitica. Cefdinir in relation to cefixime (the most active c o m p a r i s o n compound) was generally equal or slightly less active. This was especially so w h e n c o m p a r i n g the MICs0 results for the Proteae ( ~ 16-fold difference). Cefdinir and cefixime were m o r e p o t e n t t h a n cefuroxime or cefaclor, but d e m o n s t r a t e d a slightly r e d u c e d Gram-negative spectrum c o m p a r e d with the parenteral c o m p a r i s o n cephem, ceftriaxone.
Antimicrobial Activity Against GramPositive Species Table 3 illustrates MIC results for cefdinir against Gram-positive organisms. Cefdinir had the greatest
activity against oxaciUin-susceptible S. aureus (MICgo, 0.25 p,g/ml) with cefuroxime, cefaclor, ceftriaxone, and cefixime having decreased activity in that rank order. The oxacillin-resistant staphylococci were usually resistant to all cephalosporins tested. The Streptococcus pneumoniae w e r e all quite susceptible to cefdinir a n d cefixime with the exception of the penicillin-resistant strains that h a d MICs fourto 32-fold higher than penicillin-susceptible isolates. Corynebacterium jeikeium a n d all enterococci w e r e resistant to cefdinir a n d the other oral cephalosporins studied. Listed in Table 4 are cefdinir MICs for 1-3 strains of 16 rarely isolated species. The results d e m o n strate that cefdinir was minimally active against the rare Enterobacteriaceae species (7 of 14 MICs at ~12 ~g/ml) and it was inactive against all Pseudomonas spp., Achromobacter xylosoxidans, and Flavobacterium spp. Cefdinir was also m o d e s t l y active against the Bacillus cereus and Listeria monocytogenes (MIC, 4-16 ~,g/ml) isolates.
431
C e f d i n i r in v i t r o A c t i v i t y
TABLE 3
C e f d i n i r P o t e n c y A g a i n s t 272 G r a m - P o s i t i v e O r g a n i s m s C o m p a r e d w i t h F o u r O t h e r Cephalosporins MIC (p,g/ml)
Organism
No. Tested
Antimicrobial Agent
Corynebacterium jeikeium
10
Cefdinir
Enterococcus faecalis
23
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
Enterococcus spp.
3(Y
Oxacillin resistant
Oxacillin susceptible
50% 0.5
90%
Range
>16
0.06->16
> 16 >4 >32 32 >32
> 16 >4 >32 >32 >32
> 16 >4 8->32 16->32 8->32
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
4 >4 >32 16 >32
> 16 >4 >32 >32 >32
1 - > 16 >4 32->32 2->32 16->32
10
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
> 16 >4 >32 >32 >32
> 16 >4 >32 >32 >32
0.25->16 >4 1->32 2->32 16->32
22 b
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriazone
0.25 >4 1 1 2
Coagulase negative
45 c
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
0.06 >4 0.5 1 2
> 16 >4 >32 >32 >32
~0.03->16 1->4 ~32 ~0.12->32 0.5->32
Staphylococcus epidermidis
20
Cefdinir Cefixime Cefuroxime Cefaclor Ceftriaxone
~0.03 4 0.5 1 2
>16 >4 16 16 16
~16 ~0.008->4 ~0.12->32 ~0.25-16 ~32
Penicillin resistant
10e
Cefdinir Cefixime
0.12 1
2 2
Penicillin susceptible
20
Cefdinir Cefixime
0.03 0.12
0.06 0.12
~0.015-0.12 0.06-0.25
Serogroup A
22
Cefdinir Cefixime
~0.015 0.06
~0.015 0.12
~4 1 2 2
0.12-4 2->4 0.5-4 1-4 0.5-2
Staphylococcus spp.
Streptococcus pneumoniae 0.03-4 0.5-2
Streptococcus spp.
Gram-positive anaerobes
~16
0.03-0.25 ~16
432
B.M. Briggs et al.
TABLE 3
Continued
qncludes E. faecium (10 strains), E. raffinosis (5 strains), E. gallinarum (5 strains), E. casseliflavus (5 strains), E. durans (2 strains), E. avium (1 strain), and E. hirae (2 strains). Several strains were ampicilinand/or vancomycinresistant. bTen strains produced a penicillinase. qncludes S. auricularis (1 strain), S. haemolyticus (10 strains), S. sciuri (1 strain), S. hominis (9 strains), S. capitis (6 strains), S. simulans (5 strains), S. saprophyticus (4 strains), S. warneri (7 strains), and S. xylosis (2 strains). qsolates were resistant to penicillin (MICs ~0.12 ~g/ml and 1-~g oxacillindisk zones of 419 mm). qncludes Clostridium perfringens (5 strains), C. tertium (1 strain), and Peptostreptococcus spp. (8 strains). TABLE 4
In vitro Activity of Cefdinir Against 32 Strains from 16 Species
Organisms
No. Tested
MIC (~g/ml)a
Bacillus cereus Achromobacter xylosoxidans Aeromonas hydrophila Citrobacter amalonatica Enterobacter sakazakii taylorae Flavobacterium spp. Hafnia alvei Klebsiella ozaenae Listeria monocytogenes Pseudomonas cepacia fluorescens putida Salmonella typhi Serratia liquefaciens Streptococcus bovis
2 2 3 2
41, 81 >162 ~0.031, 0.122 11, 161
2 2 2 2 2 2
0.061, 21 161, >161 >162 41, 81 0.062 81, 161
2 2 2 2
> 162 >162 >162 0.061, 0.121
shown) (Eliopoulos et al., 1989). Several representative species were tested by agar dilution and broth microdilution methods (22 strains). Over 75% of results were +_1 log2 dilution step with a slight, insignificant trend toward lower MICs by the agar dilution method.
Activity Against Drug-Resistant Strains
2
22
1
~0.031
~The inferior (subscript) number is the isolated occurrence at each MIC result.
MBCs, Inoculum Effects, and Medium Influences The activity of cefdinir on the 29 selected strains was relatively unaffected by variation of inoculum concentrations with the exception of chromosomal ~lactamase-producing species (types I and IV) and the K. pneumoniae strains that produced plasmid-mediated extended-spectrum enzymes (Table 1). Neither CO2 incubation nor magnesium ion addition had an effect on cefdinir MIC values. In Table 5 the compiled data compare MICs and MBCs of cefdinir and cefixime against some ~-lactamase-producing-type bacteria. The table reflects that cefdinir has only slightly elevated MBC values (twofold) and was comparably bactericidal to cefixime. The 5- and 10-~g cefdinir disk (investigator prepared) zones were larger (~6 ram), and the MICs slightly lower in the presence of blood product supplements when testing some enterococci (data not
Cefdinir activity against various ~-lactamase-producing bacteria is summarized in Tables 1, 5, and 6. The plasmid-mediated staphylococcal penicillinases, TEM-1, TEM-2, HMS-1, OXA-1, OXA-2, OXA-3, and SHV-1 producing strains were all susceptible to cefdinir. All Pseudomonas spp. including the enzymeproducing CARB-1, CARB-2, CARB-4, and OXA-4 strains were cefdinir resistant. The MICs of organisms producing extended-spectrum ~-lactamases (Bush and Singer, 1989) are summarized in Tables 1 and 6. The cefdinir MICs for these strains with potent enzymes were generally lower than cefixime and ceftazidime. Approximately one-half of these E. coli C600 transconjugates had cefdinir MIC values that were considered susceptible (~1 ~g/ml). However, the elevated MICs of plasmid-containing strains compared with strain C600 indicates some cefdinir hydrolysis. Cefixime and ceftazidime also showed similar effects, whereas imipenem was very active against all extended-spectrum ~-lactamase-producing organisms. The activity of cefdinir against cefotaxime- and ceftazidime-resistant strains was minimal (MIC90, 16 ~,g/ml). Gentamicin-resistant, nonenteric bacilli were also cefdinir resistant with a MIC90 of 316 p,g/ml. Cefdinir had the greatest activity against gentamicin-resistant enteric bacilli (MICgo, ~2 ~g/ml). Strains with ( O m p F + ) and without ( O m p F - ) control membrane transport protein were also evaluated. Cefdinir and cefixime appear to have comparable penetration (fourfold difference in MICs), whereas imipenem maintained excellent cell access even without OmpF. The differential DC0/DC2 MIC studies also demonstrated that cefdinir had acceptable penetration into E. coli cells (Richmond et al., 1976; Richmond and Wooton, 1976). The rank order
Cefdinir in vitro Activity
TABLE 5
433
MIC a n d MBC Results for Cefdinir a n d Cefixime Cefdinir Resistance Mechanism
Organism
Escherichia coli
MIC
Type III (HMS-1) (OXA-1) (OXA-2) (OXA-3)
(SHY-l) (TEM-1) (TEM-2)
Cefixime MBC
MIC
MBC
0.25a 0.5 0.25 0.12 0.25 0.5 0.12
0.5 1 0.25 0.25 0.25 0.5 0.12
0.25 0.25 0.25 0.5 0.12 0.25 0.06
0.25 0.25 0.25 0.5 0.12 0.25 0.12
0.25 0.25
0.25 0.25
Klebsiella oxytoca
Type IV (K1) (K14)
4 4
4 8
Klebsiella pneumoniae
Type III (ExSpBL)b (ExSpBL)~
4 4
4 4
1 >4
2 >4
Pseudomonas aeruginosa
Type V (CARB-1)
16
>16
>4
>4
aConcentration in ~g/ml. bFrom Bush and Singer (1989). Abbreviations as in Table 1.
TABLE 6
Susceptibilities to Cefdinir a n d Three Currently Available (Oral and Parenteral) [3-Lactams for Escherichia coli C600 Containing Various Recently Discovered E x t e n d e d S p e c t r u m f~Lactamases a
MIC (~g/ml) ~-Lactamase Type Cefdinir Cefixime Ceftazidime Imipenem Basal strain (C600) SHV-1 SHV-2 SHV-3 SHV-4 SHV-5 TEM-1 TEM-2 TEM-3 TEM-4 TEM-5 TEM-6 TEM-7 TEM-9 CAZ-2 OmpF+ OmpF-
0.5 0.5 >16 8 >16 16 0.25 1 16 >16 16 1 1 1 4 0.5 2
1 1 >4 >4 >4 >4 0.5 1 >4 >4 >4 >4 2 >4 >4 0.25 1
0.25
0.12
2 >16 >16 >16 >16 0.25 0.5 >16 >16 >16 >16 >16 >16 >16
0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12
~< 0.12 0.25
0.12 0.12
of best to worst bacterial cell access was cefixime > cefaclor > cefdinir > cefuroxime.
DISCUSSION The introduction of a h y d r o x y i m i n o rather t h a n a c a r b o x y m e t h o x y i m i n o g r o u p as in cefixime allows e n h a n c e m e n t of cefdinir antibacterial activity against Gram-positive organisms (Mine et al., 1988). The spectrum of activity for cefdinir appears to be well balanced against Gram-positive and Gram-negative bacteria as f o u n d in this s t u d y a n d b y I n a m o t o and associates (1988). O t h e r n e w orally a d m i n i s t e r e d cephalosporins fail to inhibit Staphylococcus spp. at clinically achievable concentrations, but, in contrast, cefdinir has s h o w n some p o t e n c y against staphylococci. The signifcance of the lower cefdinir MICs for the strains with extended spectrum f~-lactamases must be evaluated in vivo (Bush a n d Singer, 1989). H i g h e r cefdinir MICs observed a m o n g the penicillin-resistant p n e u m o c o c c i were consistent with the results of Bosley et al. (1987) for other recently described cephalosporins. Again, in vivo r e s p o n s e s m u s t be closely m o n i t o r e d for these types of clinical isolates, Blood products a p p e a r to potentiate the cefdinir activity
434
against some enterococci. This p h e n o m e n o n has previously been described for several 7-methoxyimino cephems including cefotaxime and ceftriaxone (Eliopoulos et al., 1989). Because cefdinir has a m o n g the lowest cephalosporin MICs against Enterococcus spp. strains, the additional activity could prove to
B.M. Briggs et al.
be of therapeutic significance (Inamoto et al., 1988; Mine et alo, 1988; Neu et al., 1989). Thus, cefdinir appears to exhibit a promising spectrum of potential activity w h e n compared to other investigational cep h e m s currently u n d e r clinical d e v e l o p m e n t (Jones, 1988; Jones a n d Barry, 1989).
REFERENCES Bosley GS, Elliott JA, Oxtoby MJ, Facklam RR (1987) Susceptibility of relatively penicillin-resistant Streptococcus pneumoniae to newer cephalosporin antibiotics. Diagn Microbiol Infect Dis 7:21-27. Bush K, Singer SB (1989) Biochemical characteristics of extended broad spectrum beta-lactamases. Infection 17:429-433. Eliopoulos GM, Reiszner E, Willey S, Novick WJ, Moellering RC (1989) Effect of blood product medium supplements on the activity of cefotaxime and other cephalosporins against Enterococcus faecalis. Diagn Microbiol Infect Dis 12:149-156. Inamoto Y, Chiba T, Kamimura T, Takaya T (1988) FK482, a new orally active cephalosporin: synthesis and biological properties. J Antibiot 41:828-830. Jacoby GA, Carreras I (1990) Activities of ~3-1actam antibiotics against Escherichia coli strains producing extended-spectrum ~-lactamases. Antimicrob Agents Chemother 34:858-862. Jones RN (1988) Antimicrobial activity, spectrum, and pharmacokinetics of old and new orally administered cephems. Antimicrob Newsl 5:1-7. Jones RN, Barry AL (1988) Susceptibility of stably derepressed beta-lactamase producing strains to imipenem and four quinolones. Eur J Clin Microbiol 7:82-83. Jones RN, Barry AL (1989) Preliminary in vitro studies of BMY-28232: the active metabolite of the BMY-28271 cephalosporin ester. J Antimicrob Chemother 23:654-657. Jones RN, Gavan TL, Thornsberry C, et al. (1989) Standardization of disk diffusion and agar dilution susceptibility tests for Neisseria gonorrhoeae: interpretative criteria and quality control guidelines for ceftriaxone, penicillin, spectinomycin and tetracycline. J Clin Microbiol 27:2758-2766. Jorgensen JH, Redding JS, Maher LA, Howell AW (1987) Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J Clin Microbiol 25:21052113. Jorgensen HJ, Maher LA, Howell AW (1990) Use of Haemophilus test medium for broth microdilution antimi-
crobial susceptibility testing of Streptococcus pneumoniae. J Clin Microbiol 28:430-434. Mine Y, Kamimura T, Watanabe Y, et al. (1988) In vitro antibacterial activity of FK482: a new orally active cephalosporin. J Antibiot 41:1873-1889. National Committee for Clinical Laboratory Standards NCCLS (1987) Proposed guidelines M26-P: methods for determining bactericidal activity of antimicrobial agents. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1989) Tentative standard M11-T2: methods for antimicrobial susceptibility testing of anaerobic bacteria. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1990a) Approved standard M2-A4: performance standards for antimicrobial disk susceptibility tests. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1990b) Approved standard, M7-A2: standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova, PA: NCCLS. Neu HC, Saha G, Chin N-X (1989) Comparative in vitro activity and ~-lactamase stability of FK482: a new oral cephalosporin. Antimicrob Agents Chemother33:1795-1800. Pearson RD, Steigbigel RT, Davis HT, Chapman SW (1980) Method for reliable determination of minimal lethal antibiotic concentrations. Antimicrob Agents Chemother 18:699-708. Richmond MH, Wotton S (1976) Comparative study of seven cephalosporins: susceptibility to beta-lactamases and ability to penetrate the surface layers of E. coli. Antimicrob Agents Chemother 10:219-222. Richmond MH, Clark DC, Wotton S (1976) Indirect method for assessing the penetration of beta-lactamase-nonsusceptible penicillins and cephalosporins in E. coli strains. Antimicrob Agents Chemother 10:215-218. Sakamoto H, Hirose T, Nakamoto S, Hatano K, Shibayama F, Kikuchi H, Mine Y (1988) Pharmacokinetics of FK482: a new orally active cephalosporin, in animals. J Antibiot 41:1896-1905.