ANTIMcICoBIAL AGENTS AND CHzMoTHzRAPY, Dec. 1976, Copyright 0) 1976 American Society for Microbiology
Vol. 10, No. 6 Printed in U.S.A.
p. 918-920
Comparative In Vitro Studies of Cinoxacin, Nalidixic Acid, and Oxolinic Acid RALPH C. GORDON,'* LYNNE I. STEVENS, CHARLES E. EDMISTON, JR., Arm KANWAL MOHAN Departments ofHuman Development, Medicine, and Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Received for publication 23 July 1976
Cinoxacin and nalidixic acid were found to be similar in in vitro activity against 138 Shigella isolates and somewhat less active than oxolinic acid on a weight basis. Cross-resistance developed when 10 shigellae were transferred on increasing amounts of the respective agent contained in Mueller-Hinton agar. Plate dilution studies of the effect of changes in agar pH on the minimum inhibitory concentration revealed that the antibacterial activity increased with decreasing pH. Protein binding investigations revealed a high degree of binding, with nalidixic acid > oxolinic acid > cinoxacin. Cinoxacin, nalidixic acid, and oxolinic acid are synthetic organic acids that are similar in their in vitro activity against gram-negative bacteria (1, 2, 7, 8, 12). These agents are currently reserved for treatment of urinary tract infections, and cinoxacin has not yet been approved for clinical use. The present laboratory studies were carried out to compare their activity against shigellae and to investigate influence of pH on antibacterial activity, development of resistance in vitro, and the degree of binding by human serum proteins. Antimicrobial susceptibility testing was carried out on 138 isolates of Shigella (25 S. flexneri and 113 S. sonnei) using the Steers replicator (11) and incorporation of the agents into Mueller-Hinton agar (6), with a final inoculum of 104 to 105 organisms delivered to the plates. The minimum inhibitory concentration (MIC) was the lowest concentration of antimicrobial allowing growth of five or fewer colonies after 24 h at 37°C. The influence of adjustment of agar pH on MIC by the addition of 1 N NaOH or 1 N HCl was evaluated for five isolates each of S. flexneri and S. sonnei. The pH values of 7.0, 7.3, and 8.0 were measured using a pH meter at 250C and checking a portion before adding the antimicrobial. The development of bacterial resistance was investigated by transferring five isolates each of the two species on gradually increasing concentrations of the agents in agar three times per week for 6 weeks beginning with subinhibitory concentrations. MIC determinations were
then carried out on the original organisms (fresh clinical isolates) and on isolates that had been transferred on the three series of plates, looking for resistance and cross-resistance. These strains were then transferred twice a week for 3 weeks on drug-free agar, looking for loss of resistance on repeat MIC determinations. Binding by human serum was estimated using broth dilution (9) and centrifugation (5) methods with paper disk assay (10) and an antimicrobial concentration of 14 ,ug/ml of 98% serum. Results of plate dilution susceptibility studies are presented in Fig. 1. On a weight basis, 100
090 I-
Ca) 2 Ca) U.
70-
5
0 u'
*40-
~302
20r
C)
MINIMUM INHIBITING CONCENTRATION (ug/mI)
FIG. 1. In vitro susceptibility of 138 Shigella isolates to three synthetic organic antimicrobials. Symbols: A, oxolinic acid; 0, cinoxacin; O, nalidixic acid.
' Present address: Infectious Disease Research Laboratory, A. P. Beutel Health Center, Texas A & M University, College Station, TX 77843.
918
NOTrES
VOL. 10, 1976
919
TABLE 1. Effect of varying pH of Mueller-Hinton agar on MIC MIC (jLg/ml) of: Strain
Oxolinic acid
7.Oa 0.39 0.39 0.39 0.78 0.39 0.39 0.39 0.78 0.39 0.78
S. sonnei 1 S. sonnei 2 S. sonnei 3 S. sonnei 6 S. sonnei 10 S. flexneri 99 S. flexneri 113 S. flexneri 117 S. flexneri 124 S. flexneri 130 a pH of agar.
7.3 0.39 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Cinoxacin
Nalidixic acid
8.0 3.12 3.12 0.39
0.78 3.12 3.12 0.39
0.78 0.78 0.78
1-.
7.3 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25
7.0 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56
8.0 12.5 12.5 12.5 12.5 6.25 12.5 12.5 >25.0 25.0 25.0
7.0 0.78 0.78 0.78 0.78 3.12 0.78 0.78 1.56 1.56 1.56
7.3 1.56 1.56 1.56
8.0 12.5 12.5 25.0
1.56 1.56 1.56 1.56 1.56 1.56 1.56
25.0 6.25 12.5 25.0 25.0 25.0 >25.0
LU
Z-7
z
z
-
7
z 5 co
B4
Lu
z
cc
o
5
4
0
z LU 3
-J
D
-
0.1
0.2 0.4
0.8
1.6
3.2
6.3 12.5
MINIMUM INHIBITING CONCENTRATION
25 >25
(jg/ml)
FIG. 2. In vitro susceptibility of 10 Shigella isolates to oxolinic acid before and after 18 passages on agar plates containing oxolinic acid, nalidixic acid, and cinoxacin. Symbols: *, fresh clinical isolates; O, nalidixic acid transfers; 0, cinoxacin transfers; A, oxolinic acid transfers.
oxolinic acid was most active; all isolates were inhibited by 1.56 ,ug or less per ml. Cinoxacin was somewhat more active than nalidixic acid; 40% as opposed to 8% was inhibited by 1.56 ug/ ml. However, all isolates were inhibited at a concentration of 6.25 ug of either agent per ml. An increase in agar pH resulted in a raised MIC of all three agents, but this increase in MIC was less apparent with oxolinic acid (Table 1). Figures 2 through 4 illustrate that 18 passages on agar containing the single agent resulted in the development of resistance and cross-resistance of the 10 isolates. Seven of the ten strains were inhibited by 12.5 ,ug or less of oxolinic acid per ml after passage. No loss of
2
D
0~ 0.8 1.6 3.2 6.25 12.5 25 50 > 50 MINIMUM INHIBITING CONCENTRATION (mg/ml)
FIG. 3. In vitro susceptibility of 10 Shigella isolates to nalidixic acid after passage on agar plates containing nalidixic acid, cinoxacin, or oxolinic acid'. Symbols: 0, fresh clinical isolates; O nalidixic acid transfers; 0, cinoxacin transfers; A, oxolinic acid
transfers.
resistance was noted after six transfers on antimicrobial-free agar. Evaluation of the susceptibility of these Shigella isolates to other antimicrobials was not carried out in the current work; however, previous studies (3, 6) have demonstrated that nalidixic acid and oxolinic acid are active against organisms resistant to multiple other agents. The degree of serum protein binding was greater with nalidixic acid (89 to 91%) than with oxolinic acid (80 to 85%) or cinoxacin (77 to 83%). Results for nalidixic acid are in agreement with those of Buchbinder and colleagues (2) and higher than the 66% for
NOtES
920
ANTImCROB. AGENTs CHzMoTrzR. This research was supported by Public Health Service General Research Support grant RR 05668-05 from the National Institutes of Health and by a grant from Eli Lily & Co., Indianapolis, Ind. H. R. Black of the Lilly Research Laboratories furnished cinoxacin. F. C. Nachod of SterlingWinthrop Research Institute and F. J. Turner of the Warner-Lambert Research Institute kindly supplied nalidixic acid and oxolinic acid, respectively. We thank Maria Patterson for critically reviewing the manuscript.
lorn
CaM
9
m
8
m
a
(a
z
7
6
LITERATURE CITED
zc cu
5
tu
2 LU
4
m
3
m
2
m
1
lo
a1AL
v-
a
1.6 3.12 6.25 12.5 25
A
A
50 >50
MINIMUM INHIBITING CONCENTRATION (mg/ml) FIG. 4. In vitro susceptibility of 10 Shigella isolates to cinoxacin after 18 passages on agar plates containing cinoxacin, nalidixic acid, or oxolinic acid. Symbols: 0, fresh clinical isolates; 0, cinoxacin transfers; O, nalidixic acid transfers; A, oxolinic
acid transfers.
nalidixic acid and 16% for cinoxacin reported by Wick and associates (12). A recent study by Giamarellou and Jackson (4) found that increasing the concentration of horse serum in a broth dilution system had no effect on the MIC of cinoxacin when tested with Escherichia coli, Proteus, and Klebsiella-Enterobacter species. Additional work should be carried out using several methods of determination of protein binding, taking into consideration the effects of pH on antibacterial activity, to further determine the binding characteristics of these agents.
1. Atlas, E., H. Clark, F. Silverblatt, and M. Turck. 1969. Nalidixic acid and oxolinic acid in the treatment of chronic bacteriuria. Ann. Intern. Med. 70:713-721. 2. Buchbinder, M., J. C. Webb, L. Anderson, and W. R. McCabe. 1963. Laboratory studies and clinical pharmacology of nalidixic acid (WIN 18,320), p. 308-317. Antimicrob. Agents Chemother. 1962. 3.. Byers, P. A., H. L. DuPont, and M. C. Goldschmidt. 1976. Antimicrobial susceptibilities of shigellae isolated in Houston, Texas, in 1974. Antimicrob. Agents Chemother. 9:288-291. 4. Giamarellou, H., and G. G. Jackson. 1975. Antibacterial activity of cinoxacin in vitro. Antimicrob. Agents
Chemother. 7:688-692.
5. Gordon, R. C., C. Regamey, and W. M. M. Kirby. 1973. Serum protein binding of erythromycin, lincomycin and clindamycin. J. Pharm. Sci. 62:1074-1077. 6. Gordon, R. C., T. R. Thompson, W. Carlson, J. W. Dyke, and L. I. Stevens. 1975. Antimicrobial resistance of shigellae isolated in Michigan. J. Am. Med. Assoc. 231:1159-1161. 7. Kurtz, S., and M. Turck. 1975. In vitro activity of cinoxacin, an organic acid antibacterial. Antimicrob. Agents Chemother. 7:370-373. 8. Lumish, R. M., and C. W. Norden. 1975. Cinoxacin: in vitro antibacterial studies of a new organic acid. Antimicrob. Agents Chemother. 7:159-163. 9. Rolinson, G. N. 1967. The significance of protein binding of antibiotics in vitro and in vivo, p. 254-283. In A. P. Waterson (ed.), Recent advances in medical microbiology. Little, Brown & Co., Boston. 10. Simon, H. J., and E. J. Yin. 1970. Microbioassay of antimicrobial agents. Appl. Microbiol. 19:573-579. 11. Steers, E., E. L. Foltz, and B. S. Graves. 1959. Inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. (Washington, D.C.) 9:307-311. 12. Wick, W. E., D. A. Preston, W. A. White, and R. S. Gordee. 1973. Compound 64716, a new synthetic antibacterial agent. Antimicrob. Agents Chemother. 4: 415-420.
Vol. 10, No. 6 Printed in U.S.A.
p. 918-920
Comparative In Vitro Studies of Cinoxacin, Nalidixic Acid, and Oxolinic Acid RALPH C. GORDON,'* LYNNE I. STEVENS, CHARLES E. EDMISTON, JR., Arm KANWAL MOHAN Departments ofHuman Development, Medicine, and Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Received for publication 23 July 1976
Cinoxacin and nalidixic acid were found to be similar in in vitro activity against 138 Shigella isolates and somewhat less active than oxolinic acid on a weight basis. Cross-resistance developed when 10 shigellae were transferred on increasing amounts of the respective agent contained in Mueller-Hinton agar. Plate dilution studies of the effect of changes in agar pH on the minimum inhibitory concentration revealed that the antibacterial activity increased with decreasing pH. Protein binding investigations revealed a high degree of binding, with nalidixic acid > oxolinic acid > cinoxacin. Cinoxacin, nalidixic acid, and oxolinic acid are synthetic organic acids that are similar in their in vitro activity against gram-negative bacteria (1, 2, 7, 8, 12). These agents are currently reserved for treatment of urinary tract infections, and cinoxacin has not yet been approved for clinical use. The present laboratory studies were carried out to compare their activity against shigellae and to investigate influence of pH on antibacterial activity, development of resistance in vitro, and the degree of binding by human serum proteins. Antimicrobial susceptibility testing was carried out on 138 isolates of Shigella (25 S. flexneri and 113 S. sonnei) using the Steers replicator (11) and incorporation of the agents into Mueller-Hinton agar (6), with a final inoculum of 104 to 105 organisms delivered to the plates. The minimum inhibitory concentration (MIC) was the lowest concentration of antimicrobial allowing growth of five or fewer colonies after 24 h at 37°C. The influence of adjustment of agar pH on MIC by the addition of 1 N NaOH or 1 N HCl was evaluated for five isolates each of S. flexneri and S. sonnei. The pH values of 7.0, 7.3, and 8.0 were measured using a pH meter at 250C and checking a portion before adding the antimicrobial. The development of bacterial resistance was investigated by transferring five isolates each of the two species on gradually increasing concentrations of the agents in agar three times per week for 6 weeks beginning with subinhibitory concentrations. MIC determinations were
then carried out on the original organisms (fresh clinical isolates) and on isolates that had been transferred on the three series of plates, looking for resistance and cross-resistance. These strains were then transferred twice a week for 3 weeks on drug-free agar, looking for loss of resistance on repeat MIC determinations. Binding by human serum was estimated using broth dilution (9) and centrifugation (5) methods with paper disk assay (10) and an antimicrobial concentration of 14 ,ug/ml of 98% serum. Results of plate dilution susceptibility studies are presented in Fig. 1. On a weight basis, 100
090 I-
Ca) 2 Ca) U.
70-
5
0 u'
*40-
~302
20r
C)
MINIMUM INHIBITING CONCENTRATION (ug/mI)
FIG. 1. In vitro susceptibility of 138 Shigella isolates to three synthetic organic antimicrobials. Symbols: A, oxolinic acid; 0, cinoxacin; O, nalidixic acid.
' Present address: Infectious Disease Research Laboratory, A. P. Beutel Health Center, Texas A & M University, College Station, TX 77843.
918
NOTrES
VOL. 10, 1976
919
TABLE 1. Effect of varying pH of Mueller-Hinton agar on MIC MIC (jLg/ml) of: Strain
Oxolinic acid
7.Oa 0.39 0.39 0.39 0.78 0.39 0.39 0.39 0.78 0.39 0.78
S. sonnei 1 S. sonnei 2 S. sonnei 3 S. sonnei 6 S. sonnei 10 S. flexneri 99 S. flexneri 113 S. flexneri 117 S. flexneri 124 S. flexneri 130 a pH of agar.
7.3 0.39 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Cinoxacin
Nalidixic acid
8.0 3.12 3.12 0.39
0.78 3.12 3.12 0.39
0.78 0.78 0.78
1-.
7.3 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25
7.0 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56
8.0 12.5 12.5 12.5 12.5 6.25 12.5 12.5 >25.0 25.0 25.0
7.0 0.78 0.78 0.78 0.78 3.12 0.78 0.78 1.56 1.56 1.56
7.3 1.56 1.56 1.56
8.0 12.5 12.5 25.0
1.56 1.56 1.56 1.56 1.56 1.56 1.56
25.0 6.25 12.5 25.0 25.0 25.0 >25.0
LU
Z-7
z
z
-
7
z 5 co
B4
Lu
z
cc
o
5
4
0
z LU 3
-J
D
-
0.1
0.2 0.4
0.8
1.6
3.2
6.3 12.5
MINIMUM INHIBITING CONCENTRATION
25 >25
(jg/ml)
FIG. 2. In vitro susceptibility of 10 Shigella isolates to oxolinic acid before and after 18 passages on agar plates containing oxolinic acid, nalidixic acid, and cinoxacin. Symbols: *, fresh clinical isolates; O, nalidixic acid transfers; 0, cinoxacin transfers; A, oxolinic acid transfers.
oxolinic acid was most active; all isolates were inhibited by 1.56 ,ug or less per ml. Cinoxacin was somewhat more active than nalidixic acid; 40% as opposed to 8% was inhibited by 1.56 ug/ ml. However, all isolates were inhibited at a concentration of 6.25 ug of either agent per ml. An increase in agar pH resulted in a raised MIC of all three agents, but this increase in MIC was less apparent with oxolinic acid (Table 1). Figures 2 through 4 illustrate that 18 passages on agar containing the single agent resulted in the development of resistance and cross-resistance of the 10 isolates. Seven of the ten strains were inhibited by 12.5 ,ug or less of oxolinic acid per ml after passage. No loss of
2
D
0~ 0.8 1.6 3.2 6.25 12.5 25 50 > 50 MINIMUM INHIBITING CONCENTRATION (mg/ml)
FIG. 3. In vitro susceptibility of 10 Shigella isolates to nalidixic acid after passage on agar plates containing nalidixic acid, cinoxacin, or oxolinic acid'. Symbols: 0, fresh clinical isolates; O nalidixic acid transfers; 0, cinoxacin transfers; A, oxolinic acid
transfers.
resistance was noted after six transfers on antimicrobial-free agar. Evaluation of the susceptibility of these Shigella isolates to other antimicrobials was not carried out in the current work; however, previous studies (3, 6) have demonstrated that nalidixic acid and oxolinic acid are active against organisms resistant to multiple other agents. The degree of serum protein binding was greater with nalidixic acid (89 to 91%) than with oxolinic acid (80 to 85%) or cinoxacin (77 to 83%). Results for nalidixic acid are in agreement with those of Buchbinder and colleagues (2) and higher than the 66% for
NOtES
920
ANTImCROB. AGENTs CHzMoTrzR. This research was supported by Public Health Service General Research Support grant RR 05668-05 from the National Institutes of Health and by a grant from Eli Lily & Co., Indianapolis, Ind. H. R. Black of the Lilly Research Laboratories furnished cinoxacin. F. C. Nachod of SterlingWinthrop Research Institute and F. J. Turner of the Warner-Lambert Research Institute kindly supplied nalidixic acid and oxolinic acid, respectively. We thank Maria Patterson for critically reviewing the manuscript.
lorn
CaM
9
m
8
m
a
(a
z
7
6
LITERATURE CITED
zc cu
5
tu
2 LU
4
m
3
m
2
m
1
lo
a1AL
v-
a
1.6 3.12 6.25 12.5 25
A
A
50 >50
MINIMUM INHIBITING CONCENTRATION (mg/ml) FIG. 4. In vitro susceptibility of 10 Shigella isolates to cinoxacin after 18 passages on agar plates containing cinoxacin, nalidixic acid, or oxolinic acid. Symbols: 0, fresh clinical isolates; 0, cinoxacin transfers; O, nalidixic acid transfers; A, oxolinic
acid transfers.
nalidixic acid and 16% for cinoxacin reported by Wick and associates (12). A recent study by Giamarellou and Jackson (4) found that increasing the concentration of horse serum in a broth dilution system had no effect on the MIC of cinoxacin when tested with Escherichia coli, Proteus, and Klebsiella-Enterobacter species. Additional work should be carried out using several methods of determination of protein binding, taking into consideration the effects of pH on antibacterial activity, to further determine the binding characteristics of these agents.
1. Atlas, E., H. Clark, F. Silverblatt, and M. Turck. 1969. Nalidixic acid and oxolinic acid in the treatment of chronic bacteriuria. Ann. Intern. Med. 70:713-721. 2. Buchbinder, M., J. C. Webb, L. Anderson, and W. R. McCabe. 1963. Laboratory studies and clinical pharmacology of nalidixic acid (WIN 18,320), p. 308-317. Antimicrob. Agents Chemother. 1962. 3.. Byers, P. A., H. L. DuPont, and M. C. Goldschmidt. 1976. Antimicrobial susceptibilities of shigellae isolated in Houston, Texas, in 1974. Antimicrob. Agents Chemother. 9:288-291. 4. Giamarellou, H., and G. G. Jackson. 1975. Antibacterial activity of cinoxacin in vitro. Antimicrob. Agents
Chemother. 7:688-692.
5. Gordon, R. C., C. Regamey, and W. M. M. Kirby. 1973. Serum protein binding of erythromycin, lincomycin and clindamycin. J. Pharm. Sci. 62:1074-1077. 6. Gordon, R. C., T. R. Thompson, W. Carlson, J. W. Dyke, and L. I. Stevens. 1975. Antimicrobial resistance of shigellae isolated in Michigan. J. Am. Med. Assoc. 231:1159-1161. 7. Kurtz, S., and M. Turck. 1975. In vitro activity of cinoxacin, an organic acid antibacterial. Antimicrob. Agents Chemother. 7:370-373. 8. Lumish, R. M., and C. W. Norden. 1975. Cinoxacin: in vitro antibacterial studies of a new organic acid. Antimicrob. Agents Chemother. 7:159-163. 9. Rolinson, G. N. 1967. The significance of protein binding of antibiotics in vitro and in vivo, p. 254-283. In A. P. Waterson (ed.), Recent advances in medical microbiology. Little, Brown & Co., Boston. 10. Simon, H. J., and E. J. Yin. 1970. Microbioassay of antimicrobial agents. Appl. Microbiol. 19:573-579. 11. Steers, E., E. L. Foltz, and B. S. Graves. 1959. Inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. (Washington, D.C.) 9:307-311. 12. Wick, W. E., D. A. Preston, W. A. White, and R. S. Gordee. 1973. Compound 64716, a new synthetic antibacterial agent. Antimicrob. Agents Chemother. 4: 415-420.
