ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1977, p. 708-711 Copyright ©) 1977 American Society for Microbiology

Vol. 11, No. 4 Printed in U.S.A.

Cefazaflur, a New Parenteral Cephalosporin: In Vitro Studies GEORGE W. COUNTS,* DAVID GREGORY, DOLORES ZELEZNIK, AND MARVIN TURCK

Department of Medicine, University of Washington and Harborview Medical Center, Seattle, Washington 98104

Received for publication 10 December 1976

Cefazaflur was tested in vitro against 262 strains of bacteria. Inhibitory and bactericidal concentrations were determined with two inoculum sizes of bacterial cells in Mueller-Hinton broth and nutrient broth. Agar dilution studies also were performed. When tested in agar, 5.0 ,ug or less of cefazaflur per ml inhibited almost all strains of Staphylococcus aureus, Escherichia coli, Klebsiella, and Proteus mirabilis. The drug was less active against Enterobacter and indole-positive Proteus, and 7.5 ug of antibiotic per ml inhibited approximately two-thirds to one-fourth of the strains. A concentration of 50 ug of cefazaflur per ml was required for inhibition of the enterococci. There was negligible activity against Pseudomonas. The drug demonstrated less activity in broth than in agar, and a major inoculum effect was seen with some strains. For example, with a lower inoculum, 2.5 ug of cefazaflur per ml killed all strains of E. coli, whereas with the higher inoculum, 7.5 ,g of cefazaflur per ml, inhibited 64% and killed only 8% of strains. The activity of the drug for some strains was greater in Mueller-Hinton broth; for others, it was greater in nutrient broth. There were considerable differences in the results of the broth and agar studies for some species when the same medium was employed. Because of differences in activity found with different media, inocula, and method of testing, an evaluation of the eventual usefulness of cefazaflur must await the results of in vivo studies.

Cefazaflur, SKF 59962, is a new parenteral the lowest concentration of drug that produced 3 or cephalosporin containing a 7-trifluoromethyl- fewer colony-forming units on an agar plate after an 37°C. A total of 262 isolates were thioacetyl group at the 7-acyl position (3). Drug 18-h incubation at 53 including strains ofEnterobacter species, concentrations in the serum of experimental studied, which represented 27 Enterobacter cloacae, 13 Enteranimals were found to be similar to those ob- obacter aerogenes, 4 Enterobacter hafnia, 4 Enterotained with comparable doses of cephalothin, bacter agglomerans, and 5 Enterobacter liquefaciens . although cefazaflur demonstrated greater in vi- Thirty indole-positive Proteus isolates, which intro activity than cephalothin against some gen- cluded 13 Proteus morganii, 11 Proteus vulgaris, and era of organisms, including isolates of Entero- 6 Proteus rettgeri, were used. A laboratory reference strain of Escherichia coli inhibited by 1.0 ,tg of bacter (1). The purpose of this study was to evaluate the cefazaflur per ml was used as a control organism in antibacterial activity of cefazaflur in in vitro all experiments. studies utilizing several different test condiRESULTS tions. Agar dilution. The results of studies determined in agar are presented in Table 1. There MATERIALS AND METHODS Minimum inhibitory concentrations (MICs) and were no consistent differences among results minimum bactericidal concentrations (MBCs) were obtained with various species of Enterobacter determined using Mueller-Hinton (MH) broth and indole-positive Proteus isolates, and the (MHB; Difco) and nutrient broth (NB; Difco) accord- results are composited for each genus. The drug ing to previously described methods (4, 6). In broth had negligible activity against Pseudomonas dilution studies, the MBC was defined as the lowest aeruginosa, and at least 50 ,g of cefazaflur per concentration of drug yielding 10 colony-forming ml was required to inhibit more than half of units or less when about 0.005 ml of broth from each enterococcal isolates in agar. The drug was clear tube was subcultured onto agar without anti- very active against strains of E. coli, Proteus biotic. Two different inocula containing 106 and 108 viable organisms per ml were used. Agar dilution mirabilis, Klebsiella pneumoniae, and StaphyMICs were determined with Mueller-Hinton agar lococcus aureus, and 2.5 ug of cefazaflur per ml (MHA) and nutrient agar (NA). The MIC in agar, inhibited nearly all strains that had been tested obtained using a Steers replicator (5), was defined as with both inoculum sizes of bacterial cells. Ta708

EVALUATION OF CEFAZAFLUR

VOL. 1 l, 1977

709

TABLE 1. Agar dilution MICs using inocula of 1O6 and 108 organismslml in MHA and NA Inoculum Organism Enterobacter

Medium

MHA

NA Proteus, indole MHA positive NA P. mirabilis

MHA

(organisms/ ml) 10' 10'

MHA NA

Klebsiella

MHA NA

S. aureus

MHA

NA

1.0

2.5

5

7.5

10

15

20

25

50

100

250

28 9 2

57 36 15

68 53 24

73 57 32

73 60 36 26 67 27 83 40 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

75 66 40 32 80 50 93 73 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

79 66 45 32 83 53 93 77 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

81 73 49 38 90 60 93 77 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

85 75 55 47 100 90 100 83 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

87 77 77 55 100 90 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

94 85 79 79 100 10 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

10' 10'

0

0

4

15

19

10' 10' 10' 10' 10'

7 3 10 0 3 3 97 73 87 57 77 63 80 73 63 47 97 97 97 97

10 3 27 13 40 27 97 83 100 100 100 97 90 87 93 77 100 97 97 97

13 10 37 17 90 83 100 100 100 100 100 100 100 97 97 97 100 100 100 100

13 10 50 30 100 100 100 100 100 100 100 100 100 100 100 97 100 100 100 100

57 27 73 30 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

10' 10'

E. coli

50.5

2 2 2

10'

NA

Cumulative % of strains inhibited by an MIC (,ug/ml) of:

10' 10' 10' 10' 10' 10' 10' 10' 106 10'

10'f 10'

ble 1 also shows the results obtained when cefazaflur was tested in the two media. With Enterobacter strains, there was considerably more activity in MHS than in NA. For example, 15 ,ug of cefazaflur per ml inhibited approximately 70% of strains in MHA, whereas only 35% were similarly inhibited in NA. With indole-positive Proteus isolates, the drug was slightly more active in NA than in MHA,. especially when tested at lower concentrations of antibiotic. Broth dilution. Figures 1 and 2 show the results of studies that were determined in MHB and NB tested against 106 and 108 organisms/ ml. In both broth media, cefazaflur demonstrated only modest activity against Enterobacter isolates. With the lower inoculum, 250 ,ug of antibiotic per ml inhibited about half of the strains and killed only 15 to 40%. There was insignificant activity with the higher inoculum when drug concentrations of up to 250 ,ug of cefazaflur per ml were used. Results with indole-positive strains ofProteus were essentially comparable in the two media. For example, 50 jug of cefazaflur per ml was required to inhibit approximately half of the strains when tested against the lower inoculum. For strains of E. coli and Klebsiella; MBCs that had been determined with 108 inoculum were considerably higher than MICs and MBCs that had been obtained with the lower inoculum of bacterial cells. In NB, a marked disparity between inhibition and killing was observed with strains of

P. mirabilis and Klebsiella when tested with the higher inoculum. This difference in killing was less apparent in MHB. Examples of representative observations obtained when comparing cefazaflur in agar and broth media are depicted in Fig. 3. Although 50 ,g of cefazaflur per ml inhibited approximately 80% of Enterobacter strains when tested in agar, this concentration of antibiotic when studied in broth inhibited 25% at the lower inoculum, and none of the isolates was susceptible at the higher inoculum. Similarly, with isolates of indole-positive Proteus, 50 ,g of cefazaflur per ml inhibited nearly all strains which had been tested in agar at both inoculum sizes of bacterial cells. However, 50 ,g of cefazaflur per ml in a broth medium inhibited only 40% with the lower inoculum and none with the higher inoculum of bacterial cells. A difference was also observed with E. coli where MBCs were appreciably higher when the higher inoculum had been used in broth.

DISCUSSION

These in vitro studies with cefazaflur reveal that results were considerably influenced by several variables in methodology. First, the drug was very active against S. aureus and most isolates ofEnterobacteriaceae when tested in agar. Second, a significant difference was demonstrated with Enterobacter when results

710

COUNTS ET AL.

ANTIMICROB. AGENTS CHEMOTHZR.

co/i 20

2

+

401

6

S,tli,/

--

Proteus, indole positive

~ 40-

~

~

~

-

K~ ~ ~ ~ ~ ~ ~ ~ Aiebsie/l

f

40

20

p'

5 VIpyoocu

-' 260 804 60-/

~~ 40 Proteus mirotv/t~~~~~ 2.5 42.5 1.05

50 10520O25 O

100

250 1.05

Staphylococcus

ue

cureus

101520iO25 50

100

250

,ug Cefazaflur / ml

FIG. 1. Cumulative percentage of isolates inhibited (MIC) or killed (MBC) by cefazaflur tested in MHB with an inocula of 106 and 108 organisms/ml. Symbols: A, MIC of 10f organismslml; A, MBC of 10' organisms/ml; 0, MIC of 108 organisms/ml; *, MBC of 108 organisms/ml.

Z;

Q--4

4'b

A?

4

1--

't

-1

i. 1.

.1lq-l

k -t

z-

It

0

%a

Slophy/ococcus oureus

Protejs m,rofr/is

2.0 5 2.5

10 15 20 25 50

100

250

,g Cefazaflur / ml

FIG. 2. Cumulative percentage of isolates inhibited (MIC) or killed (MBC) by cefazaflur tested in NB with inocula of 106 and 108 organismslml. Symbols: A, MIC of 106 organisms/ml; A, MBC of 106 organisms/ml; 0, MIC of 108 organismslml; *, MBC of 108 organisms/ml.

EVALUATION OF CEFAZAFLUR

VOL. 11, 1977

20-

Proteus indole

100

t

S>/

0-

At

-1*

positive

MoC IE

60 §v

~~~~Escherichia7 co/i

,

2j

e

2.5

40

8

|

MHB

106 ~org /mlI

X

1.0l

5

10

15

20

25

MHA

-

4

50

100

250

p9g Cefozaflur /ml 3. Cumulative percentage of isolates of En-

FIG.

terobacter,

indole-positive Proteus, and E. coli in-

hibited (MIC)

MHB

by cefazaflur in MHA compared to

with a bacterial inocula of two different sizes

that had been obtained with one medium were compared to those which had been obtained with

another. Moreover, in MH medium, the

results were quite different when the same strains of Enterobacter were tested by agar di-

lution and not by a broth dilution technique. Third, a significant inoculum effect was demonstrated when most isolates were tested in broth. For example, with an inoculum of

106

organisms/mi the drug exerted impressive ac-

tivity,

both inhibitory and killing, for some

organisms, but it failed to inhibit or kill a significant

percentage of organisms when a higher

inoculum was employed. Of the several variables that may influence the results of in vitro studies with antibiotics, it is by no means certain which test conditions most accurately predict potential clinical effi-

cacy. Among these variables are inoculum size,

concentration of various ions, the presence absence of serum in broth testing, and testing in solid versus liquid media. A marked effect of pH on in vitro activity of 16 of 17

pH,

or

711

cephalosporins was recently reported (M. Laverdiere and L. D. Sabath, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 16th, Chicago, Ill., Abstr. 319, 1976). It was noted that those antibiotics increased their activity 1to 32-fold when the pH of the medium was decreased from 7.4 to 5.5. The initial pH of the MH medium used in our studies was 7.4 and that of nutrient was 6.8. Cefazaflur was not consistently more active in nutrient than in MH medium. There was no difference in calcium or magnesium concentrations, and we did not include serum in our studies or measure residual drug activity after incubation with organisms. The activity of cefazaflur against Enterobacter strains tested in agar was similar to results reported from our laboratory with cefamandole (2). When tested in broth, cefamandole was somewhat more active than cefazaflur or cefoxitin against these same Enterobacter isolates (2). The higher MBCs for cefazaflur found with P. mirabilis and Klebsiella strains when tested with the higher inoculum of bacterial cells in NB also were observed to some extent with both cefamandole and cefoxitin. In conclusion, cefazaflur demonstrated good antibacterial activity against many isolates of Enterobacteriaceae and S. aureus. However, because ofthe differences in activity found with different media, inocula, and method of testing, an evaluation of the eventual usefulness of cefazaflur must await the results of in vivo studies. LITERATURE CITED 1. Actor, P., J. V. Uri, J. R. Guarini, I. Zajac, L. Phillips, C. S. Sachs, R. M. DeMarinis, J. R. E. Hoover, and J. A. Weisbach. 1975. A new parenteral cephalosporin. SK&F 59962: In vitro snd in vivo antibacterial activity and serum levels in experimental animals. J. Antibiot. (Tokyo) 28:471476. 2. Adams, H. G., G. A. Stilwell, and M. Turck. 1976. In vitro evaluation of cefoxitin and cefamandole. Antimicrob. Agents Chemother. 9:1019-1024. 3. DeMarinis, R. M., J. R. E. Hoover, G. L. Dunn, P. Actor, J. V. Uri, and J. A. Weisbach. 1975. A new parenteral cephalosporin. SK&F 59962: 7-trifluoro-

methylthioacetamido-3-(1-methyl-lH-tetrazol-5-yl-

thiomethyl)-3-cephem-4-carboxylic acid. Chemistry and structure activity relationships. J. Antibiot. (Tokyo) 28:463-470. 4. Reller, L. B., W. W. Karney, H. N. Beaty, K. K. Holmes, and M. Turck. 1973. Evaluation of cefazolin, a new cephalosporin antibiotic. Antimicrob. Agents Chemother. 3:488497. 5. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 6. Turek, M., K. N. Anderson, R. H. Smith, J. F. Wallace, and R. G. Petersdorf. 1965. Laboratory and clinical evaluation of a new antibiotic-cephalothin. Ann. Intern. Med. 63:199-211.

Cefazaflur, a new parenteral cephalosporin: in vitro studies.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1977, p. 708-711 Copyright ©) 1977 American Society for Microbiology Vol. 11, No. 4 Printed in U.S.A. Ce...
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