Vol. 35, No. 3
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 497-499
0066-4804/91/030497-03$02.00/0 Copyright © 1991, American Society for Microbiology
Susceptibility of Neisseria gonorrhoeae to Cefpodoxime: Determination of MICs and Disk Diffusion Zone Diameters THOMAS FEKETE,l.2* JOHN WOODWELL,2 AND KENNETH R. CUNDY2 Departments of Internal Medicine' and Microbiology,2 Temple University Health Sciences Center, 3401 N. Broad Street, Philadelphia, Pennsylvania 19140 Received 9 October 1990/Accepted 9 January 1991
We studied the susceptibilities of 77 strains of Neisseria gonorrhoeae to four antibiotics: cefpodoxime, ceftriaxone, penicillin, and tetracycline. All strains were susceptible to ceftriaxone. Cefpodoxime MICs (range, 0.001 to 0.125 ,ug/ml) were parallel to and approximately four times those of ceftriaxone, and all strains will probably be considered susceptible to cefpodoxime. Disk diffusion zone diameters for cefpodoxime ranged from 35 to 57 mm. Of the strains, 32% were penicillin resistant and 51% were tetracycline resistant (MIC, .2 ,ug/ml). Susceptibility measurements were consistent for disk diffusion zone diameter and MIC, with an overall agreement of 215 of 225 (96%) for ceftriaxone, penicillin, and tetracycline combined. On the basis of these in vitro data, cefpodoxime should be evaluated in the treatment of gonorrhea.
rheal infection usually responds to multiple parenteral doses of ceftriaxone.
There are two major limitations in the current management of patients with gonorrhea. The first limitation is the shortage of effective antimicrobial agents which can be given in a single oral dose. Oral medications can improve acceptance of treatment, and single-dose therapy makes it unnecessary to rely on patient compliance to complete therapy at home. Quinolones (3) and cephalosporins (1) have both been useful in the oral treatment of gonorrhea. Tetracyclines (10) and penicillin (5), however, are no longer reliable therapeutic agents because of the emergence and dissemination of resistant strains of Neisseria gonorrhoeae. The second limitation is the exceptional difficulty in testing for antibiotic resistance in N. gonorrhoeae. Few clinical laboratories currently do more than test for P-lactamase production (7). The fastidious growth requirements of N. gonorrhoeae have precluded the use of the usual broth microdilution and disk diffusion technologies for routine susceptibility testing. Reference laboratories provide information about resistance patterns in different geographical regions, but this may be of limited value in treatment decisions made at the time of diagnosis. We studied the susceptibility of N. gonorrhoeae to cefpodoxime, a new oral cephalosporin. This agent is administered as cefpodoxime proxetil, an ester prodrug, which is metabolized to active drug (cefpodoxime) by intestinal esterases (13). Cefpodoxime is resistant to many bacterial P-lactamases and has an antimicrobial spectrum similar to that of cefixime but with greater activity for gram-positive organisms (9). In one in vitro study, cefpodoxime had activity superior to that of cefuroxime and comparable with that of ceftriaxone for N. gonorrhoeae (12). Cefpodoxime is well absorbed and well tolerated and has a plasma half-life of 2.7 h (2); after a single oral dose of 200 mg, levels in plasma exceed 1 ,ug/ml for more than 5 h. We compared susceptibility results for cefpodoxime, ceftriaxone, penicillin, and tetracycline. Ceftriaxone is currently the preferred therapy for the treatment of gonorrhea in the United States (4). A dose of 250 mg given intramuscularly is essentially 100% curative for gonorrhea infection of the anogenital or pharyngeal region. Disseminated gonor*
MATERIALS AND METHODS
The patient population was drawn from three clinics for sexually transmitted diseases. Two clinics in north Philadelphia that serve an urban, indigent population provided strains from 73 patients, and a clinic in San Diego provided strains from 4 patients. These 77 clinical isolates were obtained from 64 men and 13 women. Fifty-nine were from the male urethra, eight were from the cervix, five were from the throat, four were from the rectum, and one was from blood. We tested all strains for presence of P-lactamase by using the nitrocefin chromogenic assay (BBL Microbiology Systems, Cockeysville, Md.). The MICs for the four test antibiotics (penicillin, tetracycline, ceftriaxone, and cefpodoxime) were determined by using the National Committee for Clinical Laboratory Standards guidelines for agar plate dilution (11). After being identified to the species level, strains were frozen in Trypticase soy broth with 20% glycerol and maintained at -70°C. All strains were thawed simultaneously and passaged twice on chocolate agar so that vigorous growth was achieved. Trypticase soy broth was inoculated directly, and the turbidity was adjusted to 0.5 McFarland units. A further 10-fold dilution in nonsupplemented Mueller-Hinton broth was prepared and loaded into the wells of a Steers replicator. A drop of approximately 2 ,ul (104 CFU) was placed onto the surface of Gold Label GC II (single-strength) agar plates (BBL) with 1% IsoVitaleX (BBL) and 1% hemoglobin (BBL) containing serial twofold dilutions of antibiotic. Plates were incubated in 5% CO2 for 24 h at 37°C. The MIC was determined by finding the lowest concentration with no visible growth. A fine haze or a single colony was considered no growth. We also tested susceptibilities with a modification of the Kirby-Bauer technique (8) by using commercially prepared Gold Label GC II agar base with 1% IsoVitaleX but without hemoglobin (BBL). Antibiotic-impregnated disks were commercially obtained (except cefpodoxime, which was supplied by Upjohn, Kalamazoo, Mich.) and contained 10 ,ug of cefpodoxime, 30 jig of ceftriaxone, 10 U of penicillin, and 30 ,g of tetracycline. The plates were incubated for 24 h in 5% CO2 at 37°C, and zone
Corresponding author.
497
498
ANTIMICROB. AGENTS CHEMOTHER.
FEKETE ET AL. TABLE 1. Comparative activities of antimicrobial agents
Antibiotic
MIC (>g/ml)
Strains
Range
90%o
50%
0.001-0.125 0.008 0.03 Cefpodoxime All P-Lactamase positive 0.0044.06 0.008 0.06 ,B-Lactamase negative 0.001-0.125 0.008 0.03 0.001-0.03 0.002 0.008 Ceftriaxone All P-Lactamase positive 0.002-0.015 0.002 0.015 P-Lactamase negative 0.001-0.03 0.002 0.008 0.25 All 0.015-8 8 Penicillin 2-8 8 8 ,-Lactamase positive 0.008 0.03 P-Lactamase negative 0.015-4 1 >8 0.125->8 Tetracycline All 4 0.5->8 >8 ,-Lactamase positive 1 >8 ,-Lactamase negative 0.125->8
diameters were measured with calipers. Control strains were each MIC agar dilution plate and with each lot of plates used for disk diffusion. The MIC controls were WHO V (ATCC 78-063856), ATCC 49226 (F-18), ATCC 35541, and Staphylococcus aureus ATCC 29213. S. aureus ATCC 25923 was substituted for ATCC 29213 in the disk diffusion runs. run on
RESULTS
Twenty-three of the strains were ,B-lactamase positive, and the remaining 54 were ,-lactamase negative. MICs are shown in Table 1. All strains fell into the susceptible range for ceftriaxone (2 ,ug/ml. All 23 P-lactamase-producing strains were penicillin resistant, as were 2 of 54 P-lactamasenegative strains. ,-Lactamase production did not correlate with reduced susceptibility to the other antibiotics, except for tetracycline, the MIC50 of which was 1 ,ug/ml for ,-lactamase-negative strains and 4 ,ug/ml for ,-lactamase-positive strains (P = 0.025, Mann-Whitney statistic). When we examined associations among MICs of the four antibiotics, there was a strong correlation between the log MICs of cefpodoxime and ceftriaxone (Pearson r = 1.0, P < 0.001). We examined all pairwise comparisons of antibiotics, and no other significant correlation was found. Kirby-Bauer results were available for 75 strains and are shown in Table 2. Guidelines for disk diffusion susceptibility TABLE 2. Susceptibilities for N. gonorrhoeae by disk diffusion zone diametera No. of strains
Antibiotic
Ceftriaxone Penicillin
Tetracycline
Highly resistant
Moderately resistant
Susceptible
0
0 5 8
75 53 49
17 18
a Highly resistant correlates with plasmid-mediated resistance, and moderately resistant correlates with chromosomally mediated resistance. For criteria used, see reference 8.
testing of N. gonorrhoeae have been proposed for penicillin, ceftriaxone, and tetracycline (8) but have not yet been formulated for cefpodoxime. The range of zone diameters for cefpodoxime was 35 to 57 (median, 50) mm. For ceftriaxone, all strains fell into the proposed susceptible range (.35 mm). For penicillin, 17 strains had zone diameters of .19 mm (proposed cutoff for 1-lactamase positivity), 5 strains had zone diameters of between 20 and 26 mm (resistant but not P-lactamase positive), and 53 strains had zone diameters of .27 mm (susceptible or moderately susceptible). For tetracycline, 18 strains had zone diameters of
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 497-499
0066-4804/91/030497-03$02.00/0 Copyright © 1991, American Society for Microbiology
Susceptibility of Neisseria gonorrhoeae to Cefpodoxime: Determination of MICs and Disk Diffusion Zone Diameters THOMAS FEKETE,l.2* JOHN WOODWELL,2 AND KENNETH R. CUNDY2 Departments of Internal Medicine' and Microbiology,2 Temple University Health Sciences Center, 3401 N. Broad Street, Philadelphia, Pennsylvania 19140 Received 9 October 1990/Accepted 9 January 1991
We studied the susceptibilities of 77 strains of Neisseria gonorrhoeae to four antibiotics: cefpodoxime, ceftriaxone, penicillin, and tetracycline. All strains were susceptible to ceftriaxone. Cefpodoxime MICs (range, 0.001 to 0.125 ,ug/ml) were parallel to and approximately four times those of ceftriaxone, and all strains will probably be considered susceptible to cefpodoxime. Disk diffusion zone diameters for cefpodoxime ranged from 35 to 57 mm. Of the strains, 32% were penicillin resistant and 51% were tetracycline resistant (MIC, .2 ,ug/ml). Susceptibility measurements were consistent for disk diffusion zone diameter and MIC, with an overall agreement of 215 of 225 (96%) for ceftriaxone, penicillin, and tetracycline combined. On the basis of these in vitro data, cefpodoxime should be evaluated in the treatment of gonorrhea.
rheal infection usually responds to multiple parenteral doses of ceftriaxone.
There are two major limitations in the current management of patients with gonorrhea. The first limitation is the shortage of effective antimicrobial agents which can be given in a single oral dose. Oral medications can improve acceptance of treatment, and single-dose therapy makes it unnecessary to rely on patient compliance to complete therapy at home. Quinolones (3) and cephalosporins (1) have both been useful in the oral treatment of gonorrhea. Tetracyclines (10) and penicillin (5), however, are no longer reliable therapeutic agents because of the emergence and dissemination of resistant strains of Neisseria gonorrhoeae. The second limitation is the exceptional difficulty in testing for antibiotic resistance in N. gonorrhoeae. Few clinical laboratories currently do more than test for P-lactamase production (7). The fastidious growth requirements of N. gonorrhoeae have precluded the use of the usual broth microdilution and disk diffusion technologies for routine susceptibility testing. Reference laboratories provide information about resistance patterns in different geographical regions, but this may be of limited value in treatment decisions made at the time of diagnosis. We studied the susceptibility of N. gonorrhoeae to cefpodoxime, a new oral cephalosporin. This agent is administered as cefpodoxime proxetil, an ester prodrug, which is metabolized to active drug (cefpodoxime) by intestinal esterases (13). Cefpodoxime is resistant to many bacterial P-lactamases and has an antimicrobial spectrum similar to that of cefixime but with greater activity for gram-positive organisms (9). In one in vitro study, cefpodoxime had activity superior to that of cefuroxime and comparable with that of ceftriaxone for N. gonorrhoeae (12). Cefpodoxime is well absorbed and well tolerated and has a plasma half-life of 2.7 h (2); after a single oral dose of 200 mg, levels in plasma exceed 1 ,ug/ml for more than 5 h. We compared susceptibility results for cefpodoxime, ceftriaxone, penicillin, and tetracycline. Ceftriaxone is currently the preferred therapy for the treatment of gonorrhea in the United States (4). A dose of 250 mg given intramuscularly is essentially 100% curative for gonorrhea infection of the anogenital or pharyngeal region. Disseminated gonor*
MATERIALS AND METHODS
The patient population was drawn from three clinics for sexually transmitted diseases. Two clinics in north Philadelphia that serve an urban, indigent population provided strains from 73 patients, and a clinic in San Diego provided strains from 4 patients. These 77 clinical isolates were obtained from 64 men and 13 women. Fifty-nine were from the male urethra, eight were from the cervix, five were from the throat, four were from the rectum, and one was from blood. We tested all strains for presence of P-lactamase by using the nitrocefin chromogenic assay (BBL Microbiology Systems, Cockeysville, Md.). The MICs for the four test antibiotics (penicillin, tetracycline, ceftriaxone, and cefpodoxime) were determined by using the National Committee for Clinical Laboratory Standards guidelines for agar plate dilution (11). After being identified to the species level, strains were frozen in Trypticase soy broth with 20% glycerol and maintained at -70°C. All strains were thawed simultaneously and passaged twice on chocolate agar so that vigorous growth was achieved. Trypticase soy broth was inoculated directly, and the turbidity was adjusted to 0.5 McFarland units. A further 10-fold dilution in nonsupplemented Mueller-Hinton broth was prepared and loaded into the wells of a Steers replicator. A drop of approximately 2 ,ul (104 CFU) was placed onto the surface of Gold Label GC II (single-strength) agar plates (BBL) with 1% IsoVitaleX (BBL) and 1% hemoglobin (BBL) containing serial twofold dilutions of antibiotic. Plates were incubated in 5% CO2 for 24 h at 37°C. The MIC was determined by finding the lowest concentration with no visible growth. A fine haze or a single colony was considered no growth. We also tested susceptibilities with a modification of the Kirby-Bauer technique (8) by using commercially prepared Gold Label GC II agar base with 1% IsoVitaleX but without hemoglobin (BBL). Antibiotic-impregnated disks were commercially obtained (except cefpodoxime, which was supplied by Upjohn, Kalamazoo, Mich.) and contained 10 ,ug of cefpodoxime, 30 jig of ceftriaxone, 10 U of penicillin, and 30 ,g of tetracycline. The plates were incubated for 24 h in 5% CO2 at 37°C, and zone
Corresponding author.
497
498
ANTIMICROB. AGENTS CHEMOTHER.
FEKETE ET AL. TABLE 1. Comparative activities of antimicrobial agents
Antibiotic
MIC (>g/ml)
Strains
Range
90%o
50%
0.001-0.125 0.008 0.03 Cefpodoxime All P-Lactamase positive 0.0044.06 0.008 0.06 ,B-Lactamase negative 0.001-0.125 0.008 0.03 0.001-0.03 0.002 0.008 Ceftriaxone All P-Lactamase positive 0.002-0.015 0.002 0.015 P-Lactamase negative 0.001-0.03 0.002 0.008 0.25 All 0.015-8 8 Penicillin 2-8 8 8 ,-Lactamase positive 0.008 0.03 P-Lactamase negative 0.015-4 1 >8 0.125->8 Tetracycline All 4 0.5->8 >8 ,-Lactamase positive 1 >8 ,-Lactamase negative 0.125->8
diameters were measured with calipers. Control strains were each MIC agar dilution plate and with each lot of plates used for disk diffusion. The MIC controls were WHO V (ATCC 78-063856), ATCC 49226 (F-18), ATCC 35541, and Staphylococcus aureus ATCC 29213. S. aureus ATCC 25923 was substituted for ATCC 29213 in the disk diffusion runs. run on
RESULTS
Twenty-three of the strains were ,B-lactamase positive, and the remaining 54 were ,-lactamase negative. MICs are shown in Table 1. All strains fell into the susceptible range for ceftriaxone (2 ,ug/ml. All 23 P-lactamase-producing strains were penicillin resistant, as were 2 of 54 P-lactamasenegative strains. ,-Lactamase production did not correlate with reduced susceptibility to the other antibiotics, except for tetracycline, the MIC50 of which was 1 ,ug/ml for ,-lactamase-negative strains and 4 ,ug/ml for ,-lactamase-positive strains (P = 0.025, Mann-Whitney statistic). When we examined associations among MICs of the four antibiotics, there was a strong correlation between the log MICs of cefpodoxime and ceftriaxone (Pearson r = 1.0, P < 0.001). We examined all pairwise comparisons of antibiotics, and no other significant correlation was found. Kirby-Bauer results were available for 75 strains and are shown in Table 2. Guidelines for disk diffusion susceptibility TABLE 2. Susceptibilities for N. gonorrhoeae by disk diffusion zone diametera No. of strains
Antibiotic
Ceftriaxone Penicillin
Tetracycline
Highly resistant
Moderately resistant
Susceptible
0
0 5 8
75 53 49
17 18
a Highly resistant correlates with plasmid-mediated resistance, and moderately resistant correlates with chromosomally mediated resistance. For criteria used, see reference 8.
testing of N. gonorrhoeae have been proposed for penicillin, ceftriaxone, and tetracycline (8) but have not yet been formulated for cefpodoxime. The range of zone diameters for cefpodoxime was 35 to 57 (median, 50) mm. For ceftriaxone, all strains fell into the proposed susceptible range (.35 mm). For penicillin, 17 strains had zone diameters of .19 mm (proposed cutoff for 1-lactamase positivity), 5 strains had zone diameters of between 20 and 26 mm (resistant but not P-lactamase positive), and 53 strains had zone diameters of .27 mm (susceptible or moderately susceptible). For tetracycline, 18 strains had zone diameters of
