ANTimICROBIAL AGENTS
AND
CHEMoTHERAPY, Mar. 1978, p. 533-535
0066-4804/78/0013-0533$02.00/O Copyright i 1978 American Society for Microbiology
Vol. 13, No. 3
Printedin U.S.A.
NOTES Comparative In Vitro Studies of Nalidixic Acid, Oxolinic Acid, and a New Analog with Thieno (2,3-b) Pyridine Structure P. M. GILIS, A. HAEMERS,* AND S. R. PATTYN Departments of Pharmaceutical Sciences and Microbiology, University of Antwerp, B-2610 Wilrijk, Belgium
Received for publication 6 October 1977
Bacterial susceptibility to a new organic acid related to nalidixic acid and oxolinic acid was tested. The in vitro susceptibility pattem of the new compound was found to be very similar to that of nalidixic acid. In our laboratory we synthesized a series of 4- for both I and NAL, and 0.25 jug/ml for OA. One oxo-4,7-dihydrothieno (2,3-b) pyridine-5-carbox- strain of E. coli was resistant to both I and NAL ylic acids. These acids are structurally related to (MIC > 128 ,ug/ml), with a MIC of 2 lag of OA nalidixic acid (NAL) and oxolinic acid (OA), per ml. All gram-positive strains and Pseudowhich are well-known agents against gram-neg- monas aeruginosa were resistant to 64 ,ug of I or ative organisms (1-7). A preliminary bacterio- NAL per ml. All Enterococci strains were resistlogical test showed that 7-ethyl-2-methyl-4-oxo- ant to I and NAL. In general, the MICs of I and 4,7-dihydrothieno (2,3-b) pyridine-5-carboxylic NAL were similar. However, certain differences acid (I) (Fig. 1) was the most promising com- were noted. For instance, Serratia marcescens pound of the series. The present study evaluates strains were inhibited by 64 ug of I per ml, the in vitro susceptibility of different organisms whereas they were resistant to more than 128 to this new compound and to NAL and OA as ,ug of NAL per ml. Otherwise, some of the Klebwell as the potential for development of bacterial resistance to these drugs. The following 60 organisms were used in this study: 9 strains of Pseudomonas aeruginosa, 4 Klebsiella pneumoniae, 2 Proteus morganii, 2 Proteus rettgeri, 4 Staphylococcus aureus, 6 Enterococci, 2 Serratia marcescens, and 31 Escherichia coli. All strains, except one E. coli (ATCC 25922), were isolates from a clinical laboratory (Institute for Tropical Medicine, Antwerp). Antibacterial activity was determined with FIG. 1. Chemical structure of I (7-ethyl-2-methyl-4the agar-dilution method in Mueller-Hinton oxo-4,7-dihydrothieno (2,3-b) pyridine-5-carboxylic agar. Twofold dilutions in water of stock solu- acid). tions, containing 2,000 jig of the drugs per ml of 0.1 N NaOH, were added to the medium to siella sp. were more susceptible to NAL than to obtain a final concentration range from 128 to I. Without exception, the MICs of OA were 0.25 ,tg of drug per ml. A replicating device (8) lower than those of I or NAL. One K. pneumowas used. Overnight cultures of the bacteria to niae strain was resistant to both I and NAL be tested were diluted so that the inocula con- (MIC > 128 ,ug/ml); the MIC of OA was 64 tained 104 colony-forming units. The minimal itg/ml. An estimation of the bactericidal activity of inhibitory concentration (MIC) was defined as the lowest concentration of antibacterial agent the new compound and OA was made by inocallowing the growth of no more than five colonies ulating 106 colony-forming units of each of 10 at the site of inoculation after overnight incu- different strains into brain heart infusion conbation at 370C. taining the MIC of the drug and 2 drug concenResults are represented in Table 1. Proteus trations above the MIC. Subsequent inoculation sp. and E. coli were the most susceptible species, onto Mueller-Hinton agar plates and overnight with a mean MIC of 1 and 2 ug/ml, respectively, incubation resulted either in growth or no 533
534
ANTimICROB. AGENTS CHEMOTHER.
NOTES
TABLE 1. In vitro susceptibility to OA, NAL, and P NAL
OA
Organism (no. of isolates)
E. coli (31) Staphylococcus aureus (4) Proteis sp. (4)
MICa,
MIC75
0.25 1
0.25
I
MIC50
MIC75
MIC90
0.25 2
MIC90 0.25 64
2 64
2 64
4
>128
0.25
0.25
1
1
2
64 64
32 >128
128 >128
>128 >128
>128 2
>128
>128 >128
MIC50 2 128
MIC75 128
MIC90 2 >128
1
1
64 >128
>128 >128
2
0.5
(indole-positive) K. pneumoniae (4) Pseudomonas
2 16
2 64
aeruginosa (9) Enterococci (6) Serratia marcescens (2)
64 2
64 2
64 >128
>128 >128 >128 >128 >128 64 128 128 a Lowest concentration, in micrograms per milliliter, of drug inhibiting at least 50 (MIC5o), 75 (MIC75), or 90% (MIC9o) of the strains tested.
TABLE 2. Bactericidal activity of OA and I against several bacterial strains OA
Clinical
MIC
isolates0
(1&9/
E. coli
16
E. coli 40193
E. coli39945 a Institute
b MBC,
MIC
infusion MBC
ml)
g/
(pg/
(#g/
0.25 0.25 0.25 4
>128 1 2 16
1 1 1 64
8 8 8 >128
2 8 0.25
>128 16 8 1 2
64 64 2 2 2
>128 64 8 8 8
4 8 0.25 for Tropical Medicine, Antwerp.
16
0.25 0.5
TABLE 3. Effect upon MICs after a single passage of the organisms into drug-containing brain heart
I
MBCb
ml)
Proteus morganii Proteus morganii Proteus rettgeri Staphylococcus aureus Serratia marcescens K. pneumoniae E. coli39849
>128
ml)
growth. The new compound was bactericidal for a variety of gram-negative organisms (Table 2). For most of the strains tested, bactericidal activity was attained at concentrations two- to fourfold above the MIC. There was no effect with 128 ,ug of the new compound per ml against Staphylococcus aureus, whereas OA was bactericidal with a minimal bactericidal concentration of 16 ,ug/ml. The development of bacterial resistance was studied with the new compound and NAL. For three strains each of Proteus (two Proteus morganii, one Proteus rettgeri) and E. coli, inocula of 10i and 10i organisms were introduced in 5 ml of brain heart infusion containing 50 or 5 ,ug of drug per ml. Tubes showing bacterial growth were plated, and, for three colonies of each, the MIC was determined with the agar-dilution method previously used. The effects upon MICs after a single passage of the organisms into drug.containing brain heart infusion are shown in Table 3. Bacterial resistance to both agents, I
NAL MIC
(pg/mi)
Strain
ml)
Minimal bactericidal concentration.
I MIC
(Ag/ml)
Before After Before After passage passage passage passage 2 64 4 128
E. coli ATCC 25922 E. coli 39227 E. coli 4043
2
2
16 8
4
2
8 16
and NAL, was readily induced by overnight growth of a 108-organism moculum of E. coli in brain heart infusion containing 5 ,ug of the drug per ml. With a concentration of 50 or 5 ,g of I or NAL per ml, no growth was observed for Proteus sp. with an inoculum of 10i or 10i organisms, or with an inoculum of 105 organisms of E. coli. In conclusion, we think that the ultimate use of the new compound will depend on its pharmacological and toxicological properties, as well as on extended clinical studies. We thank C. Marnef and M. Borms for their excellent assistance during this study.
LITERATURE CITED 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 V. 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. Harrison, L, H., and C. E. Cox. 1970. Bacteriologic and pharmacodynamic aspects of nalidixic acid. J. Urol.
104:908-913.
4.
Kamlnsy, D., and R. I. Meltzer. 1967. Quinolone anti-
bacterial agents: oxolinic acid and related compounds. J. Med. Chem. 11:160-163. 5. Lesher, G. Y., E. J. Froelish, M. D. Gruett, J. M. Bailey, and R. P. Brundage. 1962. 1,8-Naphtyridine derivates: a new class of chemotherapeutic agents. J. Med. Pharm. Chem. 5:1063-1065.
VOL. 13, 1978 6. Ringel, S. M., F. J. Turner, F. L. Lindo, S. Roemer, B. A. Direnga, and B. S. Schwartz. 1968. Oxolinic acid, a new synthetic antimicrobial agent. II. Bactericidal rate and resistance development, p. 480485. Antimicrob. Agents Chemother. 1967. 7. Ronald, A. R., M. Turck, and R. G. Peteredorf. 1966.
NOTES
535
A critical evaluation of nalidixic acid in urinary tract infections. N. Engl. J. Med. 275:1081-1089. 8. 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.
AND
CHEMoTHERAPY, Mar. 1978, p. 533-535
0066-4804/78/0013-0533$02.00/O Copyright i 1978 American Society for Microbiology
Vol. 13, No. 3
Printedin U.S.A.
NOTES Comparative In Vitro Studies of Nalidixic Acid, Oxolinic Acid, and a New Analog with Thieno (2,3-b) Pyridine Structure P. M. GILIS, A. HAEMERS,* AND S. R. PATTYN Departments of Pharmaceutical Sciences and Microbiology, University of Antwerp, B-2610 Wilrijk, Belgium
Received for publication 6 October 1977
Bacterial susceptibility to a new organic acid related to nalidixic acid and oxolinic acid was tested. The in vitro susceptibility pattem of the new compound was found to be very similar to that of nalidixic acid. In our laboratory we synthesized a series of 4- for both I and NAL, and 0.25 jug/ml for OA. One oxo-4,7-dihydrothieno (2,3-b) pyridine-5-carbox- strain of E. coli was resistant to both I and NAL ylic acids. These acids are structurally related to (MIC > 128 ,ug/ml), with a MIC of 2 lag of OA nalidixic acid (NAL) and oxolinic acid (OA), per ml. All gram-positive strains and Pseudowhich are well-known agents against gram-neg- monas aeruginosa were resistant to 64 ,ug of I or ative organisms (1-7). A preliminary bacterio- NAL per ml. All Enterococci strains were resistlogical test showed that 7-ethyl-2-methyl-4-oxo- ant to I and NAL. In general, the MICs of I and 4,7-dihydrothieno (2,3-b) pyridine-5-carboxylic NAL were similar. However, certain differences acid (I) (Fig. 1) was the most promising com- were noted. For instance, Serratia marcescens pound of the series. The present study evaluates strains were inhibited by 64 ug of I per ml, the in vitro susceptibility of different organisms whereas they were resistant to more than 128 to this new compound and to NAL and OA as ,ug of NAL per ml. Otherwise, some of the Klebwell as the potential for development of bacterial resistance to these drugs. The following 60 organisms were used in this study: 9 strains of Pseudomonas aeruginosa, 4 Klebsiella pneumoniae, 2 Proteus morganii, 2 Proteus rettgeri, 4 Staphylococcus aureus, 6 Enterococci, 2 Serratia marcescens, and 31 Escherichia coli. All strains, except one E. coli (ATCC 25922), were isolates from a clinical laboratory (Institute for Tropical Medicine, Antwerp). Antibacterial activity was determined with FIG. 1. Chemical structure of I (7-ethyl-2-methyl-4the agar-dilution method in Mueller-Hinton oxo-4,7-dihydrothieno (2,3-b) pyridine-5-carboxylic agar. Twofold dilutions in water of stock solu- acid). tions, containing 2,000 jig of the drugs per ml of 0.1 N NaOH, were added to the medium to siella sp. were more susceptible to NAL than to obtain a final concentration range from 128 to I. Without exception, the MICs of OA were 0.25 ,tg of drug per ml. A replicating device (8) lower than those of I or NAL. One K. pneumowas used. Overnight cultures of the bacteria to niae strain was resistant to both I and NAL be tested were diluted so that the inocula con- (MIC > 128 ,ug/ml); the MIC of OA was 64 tained 104 colony-forming units. The minimal itg/ml. An estimation of the bactericidal activity of inhibitory concentration (MIC) was defined as the lowest concentration of antibacterial agent the new compound and OA was made by inocallowing the growth of no more than five colonies ulating 106 colony-forming units of each of 10 at the site of inoculation after overnight incu- different strains into brain heart infusion conbation at 370C. taining the MIC of the drug and 2 drug concenResults are represented in Table 1. Proteus trations above the MIC. Subsequent inoculation sp. and E. coli were the most susceptible species, onto Mueller-Hinton agar plates and overnight with a mean MIC of 1 and 2 ug/ml, respectively, incubation resulted either in growth or no 533
534
ANTimICROB. AGENTS CHEMOTHER.
NOTES
TABLE 1. In vitro susceptibility to OA, NAL, and P NAL
OA
Organism (no. of isolates)
E. coli (31) Staphylococcus aureus (4) Proteis sp. (4)
MICa,
MIC75
0.25 1
0.25
I
MIC50
MIC75
MIC90
0.25 2
MIC90 0.25 64
2 64
2 64
4
>128
0.25
0.25
1
1
2
64 64
32 >128
128 >128
>128 >128
>128 2
>128
>128 >128
MIC50 2 128
MIC75 128
MIC90 2 >128
1
1
64 >128
>128 >128
2
0.5
(indole-positive) K. pneumoniae (4) Pseudomonas
2 16
2 64
aeruginosa (9) Enterococci (6) Serratia marcescens (2)
64 2
64 2
64 >128
>128 >128 >128 >128 >128 64 128 128 a Lowest concentration, in micrograms per milliliter, of drug inhibiting at least 50 (MIC5o), 75 (MIC75), or 90% (MIC9o) of the strains tested.
TABLE 2. Bactericidal activity of OA and I against several bacterial strains OA
Clinical
MIC
isolates0
(1&9/
E. coli
16
E. coli 40193
E. coli39945 a Institute
b MBC,
MIC
infusion MBC
ml)
g/
(pg/
(#g/
0.25 0.25 0.25 4
>128 1 2 16
1 1 1 64
8 8 8 >128
2 8 0.25
>128 16 8 1 2
64 64 2 2 2
>128 64 8 8 8
4 8 0.25 for Tropical Medicine, Antwerp.
16
0.25 0.5
TABLE 3. Effect upon MICs after a single passage of the organisms into drug-containing brain heart
I
MBCb
ml)
Proteus morganii Proteus morganii Proteus rettgeri Staphylococcus aureus Serratia marcescens K. pneumoniae E. coli39849
>128
ml)
growth. The new compound was bactericidal for a variety of gram-negative organisms (Table 2). For most of the strains tested, bactericidal activity was attained at concentrations two- to fourfold above the MIC. There was no effect with 128 ,ug of the new compound per ml against Staphylococcus aureus, whereas OA was bactericidal with a minimal bactericidal concentration of 16 ,ug/ml. The development of bacterial resistance was studied with the new compound and NAL. For three strains each of Proteus (two Proteus morganii, one Proteus rettgeri) and E. coli, inocula of 10i and 10i organisms were introduced in 5 ml of brain heart infusion containing 50 or 5 ,ug of drug per ml. Tubes showing bacterial growth were plated, and, for three colonies of each, the MIC was determined with the agar-dilution method previously used. The effects upon MICs after a single passage of the organisms into drug.containing brain heart infusion are shown in Table 3. Bacterial resistance to both agents, I
NAL MIC
(pg/mi)
Strain
ml)
Minimal bactericidal concentration.
I MIC
(Ag/ml)
Before After Before After passage passage passage passage 2 64 4 128
E. coli ATCC 25922 E. coli 39227 E. coli 4043
2
2
16 8
4
2
8 16
and NAL, was readily induced by overnight growth of a 108-organism moculum of E. coli in brain heart infusion containing 5 ,ug of the drug per ml. With a concentration of 50 or 5 ,g of I or NAL per ml, no growth was observed for Proteus sp. with an inoculum of 10i or 10i organisms, or with an inoculum of 105 organisms of E. coli. In conclusion, we think that the ultimate use of the new compound will depend on its pharmacological and toxicological properties, as well as on extended clinical studies. We thank C. Marnef and M. Borms for their excellent assistance during this study.
LITERATURE CITED 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 V. 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. Harrison, L, H., and C. E. Cox. 1970. Bacteriologic and pharmacodynamic aspects of nalidixic acid. J. Urol.
104:908-913.
4.
Kamlnsy, D., and R. I. Meltzer. 1967. Quinolone anti-
bacterial agents: oxolinic acid and related compounds. J. Med. Chem. 11:160-163. 5. Lesher, G. Y., E. J. Froelish, M. D. Gruett, J. M. Bailey, and R. P. Brundage. 1962. 1,8-Naphtyridine derivates: a new class of chemotherapeutic agents. J. Med. Pharm. Chem. 5:1063-1065.
VOL. 13, 1978 6. Ringel, S. M., F. J. Turner, F. L. Lindo, S. Roemer, B. A. Direnga, and B. S. Schwartz. 1968. Oxolinic acid, a new synthetic antimicrobial agent. II. Bactericidal rate and resistance development, p. 480485. Antimicrob. Agents Chemother. 1967. 7. Ronald, A. R., M. Turck, and R. G. Peteredorf. 1966.
NOTES
535
A critical evaluation of nalidixic acid in urinary tract infections. N. Engl. J. Med. 275:1081-1089. 8. 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.
