ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1978, p. 45-50 0066-4804/78/0014-0045$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 14, No. 1 Printed in U.S.A.
Differences in Susceptibilities of Virulent Strains and Avirulent Strains of Neisseria gonorrhoeae to Antimicrobial Agents DANIEL V. LIM University of South Florida, Tampa, Florida 33620 Department of Biology,
Received for publication 8 December 1978
Virulent cells (Ti colony type) and avirulent cells (T4 colony type) of Neisseria gonorrhoeae were grown in a defined liquid medium in the presence of different antimicrobial agents. Bacteria of Ti colony type were found to be more resistant than bacteria of T4 colony type to the inhibitory effects of specific concentrations of ampicillin (0.50 ytg/ml) and penicillin (0.01 ag/ml). Bacteria of T4 colony type, however, were found to be more resistant to the effects of chloramphenicol (0.40 Itg/ml), erythromycin (0.10 ,ug/ml), spectinomycin (10.00 tig/ml), and tetracycline (0.30 ,tg/ml). The differences in susceptibilities of these bacteria to the antibiotics investigated were not due to differences in growth rate or to differences in permeability. The data suggest that virulent gonococci and avirulent gonococci have unique properties that affect their susceptibilities to certain antimicrobial agents.
rhoeae to six antimicrobial agents (ampicillin, chloramphenicol, erythromycin, penicillin, spectinomycin, and tetracycline). MATERIALS AND METHODS Bacteria. N. gonorrhoeae strain F.62 was furnished by D. S. Kellogg, Jr., Center for Disease Control,
Gonococcal infections in men and women routinely are treated with antimicrobial agents such as penicillin, ampicillin, tetracycline, or spectinomycin (26). The quantities of these antibiotics used in therapy, however, has increased over the years as their effectiveness in treatment of gonorrhea has diminished (13, 24, 28). Recently, with the emergence of penicillin-resistant (1, 2, 16) and spectinomycin-resistant (1) strains of Neisseria gonorrhoeae, there has been increased concern that treatment of uncomplicated gonococcal infections with these antibiotics eventually may not be sufficient. Although previous studies have shown that antibiotic resistance may result from plasmidmediated resistance genes (2) or from permeability barriers in the cell (20, 22) and that multiple antibiotic resistance is derived from clusters of linked genes on bacterial chromosomes (21, 25), there have been few reports comparing susceptibilities of virulent strains and avirulent strains of N. gonorrhoeae to antimicrobial agents. Such data could provide valuable information on metabolic and morphological differences in these strains of gonococci, as well as an insight into the basis of antibiotic resistance in
Atlanta, Ga. Clinical strains were obtained from the Tampa Regional Laboratory, Department of Health and Rehabilitative Services, Tampa, Fla. Colony types Ti and T4 were selected from these strains, and cultures of the bacteria were stored at -80°C in Trypticase soy broth (Baltimore Biological Laboratory [BBL], Cockeysville, Md.) with 20% (vol/vol) glycerol. Bacteria of Ti and T4 colony types were grown on plates of GC agar base (BBL) supplemented with 1% (vol/vol) IsoVitaleX (BBL) and maintained by daily transfer on this medium in 5% CO2 at 36°C in a waterjacketed C02 incubator (model 3156, Forma Scientific, Marietta, Ohio). We refer to this medium as GCB. Growth media. A variation of the chemically defined medium of Wood and Brownell (30) was used for growth of the bacteria. The modified chemically defined medium (MCDM) contained uracil and hypoxanthine at final concentrations of 0.355 and 0.294 mM, respectively. In addition, 10 mM glycerol, 1.3 mM sodium lactate, 0.125% pyruvic acid, 1.52 mM oxalacetic acid, and 0.004 mM calcium pantothenate were added to the medium. Liquid medium and solid medium were identical with the exception that the solid medium also contained purified agar (Difco Laboratories, Detroit, Mich.) at a final concentration of 1% (wt/vol).
these bacteria. In this study we describe differences in susceptibilities of virulent (Ti colony type) and avirulent (T4 colony type) strains of N. gonor45
46
ANTIMICROB. AGENTS CHEMOTHER.
LIM
Growth of bacteria. Bacteria of Ti and T4 colony types, after one transfer on plates of solid MCDM incubated in 5% CO2, were inoculated into liquid MCDM (6-ml liquid volume in a 50-ml Erlenmeyer flask). The initial absorbance of the culture was 0.100 optical density unit (equivalent to 107 colony-formiing units [CFU] per ml of medium), as measured at a wavelength of 520 nm on a DB-GT grating spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). The bacteria were incubated at 36°C in a reciprocal water bath shaker at a setting of 80 complete 1-inch (ca. 2.54-cm) reciprocal strokes per min. Antimicrobial agents were dissolved in sterile distilled water and filter-sterilzed through a 0.22-,um filter before addition to cell suspensions. Sample cultures were removed from the flasks at intervals, mixed on a Vortex-Genie mixer (Scientific Industries, Inc., Bohemia, N.Y.), and either read for absorbance dn a spectrophotometer or plated onto GCB to determine viable count. Viable counts were determined by dilution of bacterial samples in dilution fluid containing 10 g of proteose peptone (Difco Laboratories) and 8.5 g of NaCl per liter of distilled water. Because piliated gonococci have a tendency to clUmp when grown in liquid culture, sanples of bactetia with Ti colony type were mixed Pbr i min whereas bacteria of T4 colony type were mlixed for 15 s on a Vortex-Genie mixer to disperse these clumps. The bacteria were plated onto GCB, and the number of CFU on these plates was determined as described previously (11). Materials. All chemicals were of reagent grade; unless otherwise indicated. Chloramphenicol, erythromycin, penicillin G, and tetracycline were putchased from Sigma Chemical Co., St. Louis, Mo., and afnipicillin and spectinomycin were purchased fronm The Upjohn Co., Kalamazoo, Mich.
RESULTS Growth of strain F62 in the presence of antimicrobial agents. Cultures of N. gonorrhoeae F62, colony types Ti and T4, grown in liquid MCDM, had different degrees of susceptibility to antimicrobial agents. Bacteria of Ti colony type were more resstant than bacteria of T4 colony type to the bactericidal effects of ampicillin and penicillin. In the presence of 0.50 ,ug of ampicillin per ml or 0.01 ,g of penicillin per ml, added to cultures at the commencement of incubation, virulent gonococci (Ti colony type) grew to higher cell densities than avirulent gonococci (T4 colony type), as measured by absorbance readings on a spectrophotometer (Fig. 1) and by the total number of CFU on plates of GCB (Table 1). In contrast, bacteria of T4 colony type were more resistant to the effects of chloramphenicol, erythromycin, spectinomycin, and tetracycline than bacteria of Ti colony type. The resistance of the bacteria to these antimicrobial agents, as
measured by absorbance readings (Fig. 1) and CFU (Table 1) after 6 h of incubation, was greatest at specific concentrations of chloramphenicol (0.4 Ag/ml), erythromycin (0.1 ,ug/ml), spectinomycin (10.0 ,ug/ml), and tetracycline (0.3
pug/ml).
The concentration of each antimicrobial agent present in cultures in these experiments was important, because the degree of difference in susceptibilities between the two colony types varied with the concentration of antibiotics used. For example, there was little or no difference in growth of virulent and avirulent gonococci in the presence of 0.001, 0.005, and 1.000 pig of penicillin per ml. However, at concentrations of penicillin between 0.005 and 1.000 pg/ml, cultures of virulent gonococci reached an absorbance reading 50% greater than that of avirulent gonococcal cultures after 6 h of incubation. Similarly, differences in growth of Ti and T4 cultures were small when the concentrations of erythromycin used were other than 0.10 pg/ml. Comparable data dependent on antibiotic concentration were obtained with each of the six antimicrobial agents investigated in this study. Because gonococci of Ti and T4 colony types have different rates of growth (11), the possibility existed that susceptibility to these antimicrobial agents may have been a function of cell growth rather than of colony type. To eliminate this possibility, Ti cultures and T4 cultures were grown to similar cell densities (3.4 x 107 CFU/ml) before the addition of ampicillin or penicillin. The data obtained (Table 2) showed that, although the T4 culture grew at a faster rate than the Ti culture, both virulent and avirulent gonococci had antibiotic susceptibilities similar to the data presented in the previous studies. Growth of elinical strains in the presence of antimicrobial agents. Bacteria of Ti and T4 colony types from several clinical strains were grown in the presence of the six antimicrobial agents. These strains had patterns of sus-
ceptibilities
to
chloramphenicol, erythromycin,
spectinomycin, and tetracycline similar to the patterns observed with strain F62. However, dif-
ferences in susceptibilities of Ti cultures and T4 cultures of these clinical strains to ampicillin and penicillin occurred only at higher concentrations of these antimicrobial agents. As shown in Table 3, Ti cultures of clinical strain USF2, a male urethral isolate, were more resistant than T4 cultures to the effects of ampicillin and penicillin at concentrations of 5.0 and 0.25 ug/ml, respectively, than at the lower concentrations of
) and T4 cultures (-----) in the presence of different FIG. 1. Growth of N. gonorrhoeae F62 Tl cultures ( antimicrobial agents. Deviations from the mean value obtained in three separate experiments are shown for each concentration point.
AMPICILLIN
P
100 80 60
-
40
-
20
-
0.001 100, 80 7
0.1 0.1
0.01
CHLORAMPHENICOL
1.0
10.0
TETRACYCLINE
IN.
60 _ 40 -1
20-i
4
0.1
5
1.0
1.0
10.0 0.1
10.0
ERYTHROMYCIN
100.
80. 60
40.
20.
0.01
0.1
1.0
10.0
100.0
SPECTINOMYCIN
100 80_
60_ 40_ 20-
1.0 CONCENTRATION (pg/mI)
47
100.0
48
ANTIMICROB. AGENTS CHEMOTHER.
LIM
TABLE 1. Growth of N. gonorrhoeae F62 in the presence of different antibiotiCsa Antibiotc
Concn
(ntlblotLc Xg/m1)
Ti
culture
CFU/ml (% of control cultureb)
T4 culture
1.22 x 109 1.12 X 109 8.17 X 104 (0.007 ± 0.005) 5.75 x 105 (0.052 ± 0.017) 0.50 3.42 x 105 (0.028 ± 0.021) 0.01 4.82 x 106 (0.430 ± 0.182) PeniciNlin 3.21 x 10" (26.349 ± 2.857) 0.40 1.49 x 10" (13.289 ± 1.163) Chloramphenicol 3.55 x 108 (29.121 ± 2.747) 0.10 8.48 X 107 (7.569 ± 0.413) Erythromycin 2.64 x 107 (2.166 ± 0.510) 8.85 x 106 (0.790 ± 0.264) 10.00 Spectinomycin 2.84 x 10" (23.267 ± 0.759) 0.30 9.69 x 107 (8.653 ± 2.367) Tetracychne a Bacteria of Ti and T4 colony types were grown in liquid MCDM at 36°C in a water bath shaker, as described in the text. After 6 h of incubation, samples were plated onto plates of GCB. The plates were incubated for 24 h at 36°C in 5% C02, and the CFU were counted. Samples from Ti cultures contained 90% Ti colony types and 10% T4 colony types, whereas samples from T4 cultures contained 100% T4 colony types. Data presented represent total CFU on the plates. Control cultures contained no antibiotics. b Values represent the mean and range of three separate experiments.
None AmpiciUlin
these antimicrobial agents used in studies with strain F62. The differences observed also were not as great as with the laboratory strain. DISCUSSION Since the introduction of penicillin as the antimicrobial agent of choice for the treatment of uncomplicated gonococcal infections, the resistance of N. gonorrhoeae to penicillin has increased steadily (13). In recent years gonococcal strains containing a plasmid-mediated beta-lactamase gene that degrades the beta-lactam ring of penicillin and makes the antibiotic ineffective in chemotherapy have been isolated in Great Britain (16), the Far East (15), and the United States (1). Other clinical isolates have been found to be partially or totally resistant to am-
picillin (28), spectinomycin (10), tetracycline (28), and other antimicrobial agents (3, 14, 23, 26). These antibiotic-resistant strains present a problem in management of gonorrhea and emphasize the need for further understanding of the mechanisms involved in antibiotic resistance.
Previous studies in this laboratory have shown that virulent (Ti colony type) and avirulent (T4 colony type) strains of N. gonorrhoeae have different rates of growth and metabolism (11). Other investigators have noted other differences in the two colonial types, such as morphology on solid medium (7, 9, 19), degree of piliation (8, 18), cell wall components (6, 17, 27, 29), and resistance to the bactericidal effects of serum (12). In this report we present evidence that virulent and avirulent strains of gonococci differ in their susceptibilities to antimicrobial agents. Our results show that bacteria of Ti and T4 colony types of N. gonorrhoeae have different degrees of susceptibility to antimicrobial agents. Ti cultures of laboratory strain F62 were more resistant than T4 cultures to the effects of ampicillin and penicillin, whereas T4 cultures were
more resistant to the effects of chloramphenicol, erythromycin, spectinomycin, and tetracycline. These differences in susceptibility could not be attributed to differences in growth rates of the two colony types, because similar results were obtained from cultures of both colony types grown for identical periods of time (Table 2). Both types of bacteria also were equally permeable to the uptake of crystal violet (data not shown). As demonstrated by Gustafsson et al. (4) and Guymon and Sparling (5), uptake of crystal violet is representative of the permeability of bacterial cells to antibiotics. Thus, decreases in susceptibility to antibiotics were not the result of decreased permeability to these
agents.
Susceptibility of Ti and T4 cultures to different antimicrobial agents, however, was dependent on the concentration of the particular agent used. Differences in susceptibilities of bacteria representing the two colony types to antimicrobial agents were greatest only at specific concentrations of the antibiotics investigated and negligible at other concentrations. This observation may explain why other investigators, using only single concentrations of antibiotics, have noted very little difference in susceptibilities of virulent and avirulent gonococci (7, 9). Clinical isolates of N. gonorrhoeae differed from laboratory strain F62 in their susceptibilities to ampicillin and penicillin. Differences in susceptibilities of clinical strains to ampicillin and penicillin occurred only at higher concentrations of the antibiotics. The extent of these differences also was greatly reduced as compared with strain F62. These observations indicated that the clinical strains, passaged only four to five times on artificial media, had developed an increased resistance to ampicillin and penicillin, as compared with strain F62, which has been passaged extensively (at least 2,500 times) on artificial media (9).
SUSCEPTIBILITY OF GONOCOCCI TO ANTIBIOTICS
VOL. 14, 1978
00
Vol. 14, No. 1 Printed in U.S.A.
Differences in Susceptibilities of Virulent Strains and Avirulent Strains of Neisseria gonorrhoeae to Antimicrobial Agents DANIEL V. LIM University of South Florida, Tampa, Florida 33620 Department of Biology,
Received for publication 8 December 1978
Virulent cells (Ti colony type) and avirulent cells (T4 colony type) of Neisseria gonorrhoeae were grown in a defined liquid medium in the presence of different antimicrobial agents. Bacteria of Ti colony type were found to be more resistant than bacteria of T4 colony type to the inhibitory effects of specific concentrations of ampicillin (0.50 ytg/ml) and penicillin (0.01 ag/ml). Bacteria of T4 colony type, however, were found to be more resistant to the effects of chloramphenicol (0.40 Itg/ml), erythromycin (0.10 ,ug/ml), spectinomycin (10.00 tig/ml), and tetracycline (0.30 ,tg/ml). The differences in susceptibilities of these bacteria to the antibiotics investigated were not due to differences in growth rate or to differences in permeability. The data suggest that virulent gonococci and avirulent gonococci have unique properties that affect their susceptibilities to certain antimicrobial agents.
rhoeae to six antimicrobial agents (ampicillin, chloramphenicol, erythromycin, penicillin, spectinomycin, and tetracycline). MATERIALS AND METHODS Bacteria. N. gonorrhoeae strain F.62 was furnished by D. S. Kellogg, Jr., Center for Disease Control,
Gonococcal infections in men and women routinely are treated with antimicrobial agents such as penicillin, ampicillin, tetracycline, or spectinomycin (26). The quantities of these antibiotics used in therapy, however, has increased over the years as their effectiveness in treatment of gonorrhea has diminished (13, 24, 28). Recently, with the emergence of penicillin-resistant (1, 2, 16) and spectinomycin-resistant (1) strains of Neisseria gonorrhoeae, there has been increased concern that treatment of uncomplicated gonococcal infections with these antibiotics eventually may not be sufficient. Although previous studies have shown that antibiotic resistance may result from plasmidmediated resistance genes (2) or from permeability barriers in the cell (20, 22) and that multiple antibiotic resistance is derived from clusters of linked genes on bacterial chromosomes (21, 25), there have been few reports comparing susceptibilities of virulent strains and avirulent strains of N. gonorrhoeae to antimicrobial agents. Such data could provide valuable information on metabolic and morphological differences in these strains of gonococci, as well as an insight into the basis of antibiotic resistance in
Atlanta, Ga. Clinical strains were obtained from the Tampa Regional Laboratory, Department of Health and Rehabilitative Services, Tampa, Fla. Colony types Ti and T4 were selected from these strains, and cultures of the bacteria were stored at -80°C in Trypticase soy broth (Baltimore Biological Laboratory [BBL], Cockeysville, Md.) with 20% (vol/vol) glycerol. Bacteria of Ti and T4 colony types were grown on plates of GC agar base (BBL) supplemented with 1% (vol/vol) IsoVitaleX (BBL) and maintained by daily transfer on this medium in 5% CO2 at 36°C in a waterjacketed C02 incubator (model 3156, Forma Scientific, Marietta, Ohio). We refer to this medium as GCB. Growth media. A variation of the chemically defined medium of Wood and Brownell (30) was used for growth of the bacteria. The modified chemically defined medium (MCDM) contained uracil and hypoxanthine at final concentrations of 0.355 and 0.294 mM, respectively. In addition, 10 mM glycerol, 1.3 mM sodium lactate, 0.125% pyruvic acid, 1.52 mM oxalacetic acid, and 0.004 mM calcium pantothenate were added to the medium. Liquid medium and solid medium were identical with the exception that the solid medium also contained purified agar (Difco Laboratories, Detroit, Mich.) at a final concentration of 1% (wt/vol).
these bacteria. In this study we describe differences in susceptibilities of virulent (Ti colony type) and avirulent (T4 colony type) strains of N. gonor45
46
ANTIMICROB. AGENTS CHEMOTHER.
LIM
Growth of bacteria. Bacteria of Ti and T4 colony types, after one transfer on plates of solid MCDM incubated in 5% CO2, were inoculated into liquid MCDM (6-ml liquid volume in a 50-ml Erlenmeyer flask). The initial absorbance of the culture was 0.100 optical density unit (equivalent to 107 colony-formiing units [CFU] per ml of medium), as measured at a wavelength of 520 nm on a DB-GT grating spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). The bacteria were incubated at 36°C in a reciprocal water bath shaker at a setting of 80 complete 1-inch (ca. 2.54-cm) reciprocal strokes per min. Antimicrobial agents were dissolved in sterile distilled water and filter-sterilzed through a 0.22-,um filter before addition to cell suspensions. Sample cultures were removed from the flasks at intervals, mixed on a Vortex-Genie mixer (Scientific Industries, Inc., Bohemia, N.Y.), and either read for absorbance dn a spectrophotometer or plated onto GCB to determine viable count. Viable counts were determined by dilution of bacterial samples in dilution fluid containing 10 g of proteose peptone (Difco Laboratories) and 8.5 g of NaCl per liter of distilled water. Because piliated gonococci have a tendency to clUmp when grown in liquid culture, sanples of bactetia with Ti colony type were mixed Pbr i min whereas bacteria of T4 colony type were mlixed for 15 s on a Vortex-Genie mixer to disperse these clumps. The bacteria were plated onto GCB, and the number of CFU on these plates was determined as described previously (11). Materials. All chemicals were of reagent grade; unless otherwise indicated. Chloramphenicol, erythromycin, penicillin G, and tetracycline were putchased from Sigma Chemical Co., St. Louis, Mo., and afnipicillin and spectinomycin were purchased fronm The Upjohn Co., Kalamazoo, Mich.
RESULTS Growth of strain F62 in the presence of antimicrobial agents. Cultures of N. gonorrhoeae F62, colony types Ti and T4, grown in liquid MCDM, had different degrees of susceptibility to antimicrobial agents. Bacteria of Ti colony type were more resstant than bacteria of T4 colony type to the bactericidal effects of ampicillin and penicillin. In the presence of 0.50 ,ug of ampicillin per ml or 0.01 ,g of penicillin per ml, added to cultures at the commencement of incubation, virulent gonococci (Ti colony type) grew to higher cell densities than avirulent gonococci (T4 colony type), as measured by absorbance readings on a spectrophotometer (Fig. 1) and by the total number of CFU on plates of GCB (Table 1). In contrast, bacteria of T4 colony type were more resistant to the effects of chloramphenicol, erythromycin, spectinomycin, and tetracycline than bacteria of Ti colony type. The resistance of the bacteria to these antimicrobial agents, as
measured by absorbance readings (Fig. 1) and CFU (Table 1) after 6 h of incubation, was greatest at specific concentrations of chloramphenicol (0.4 Ag/ml), erythromycin (0.1 ,ug/ml), spectinomycin (10.0 ,ug/ml), and tetracycline (0.3
pug/ml).
The concentration of each antimicrobial agent present in cultures in these experiments was important, because the degree of difference in susceptibilities between the two colony types varied with the concentration of antibiotics used. For example, there was little or no difference in growth of virulent and avirulent gonococci in the presence of 0.001, 0.005, and 1.000 pig of penicillin per ml. However, at concentrations of penicillin between 0.005 and 1.000 pg/ml, cultures of virulent gonococci reached an absorbance reading 50% greater than that of avirulent gonococcal cultures after 6 h of incubation. Similarly, differences in growth of Ti and T4 cultures were small when the concentrations of erythromycin used were other than 0.10 pg/ml. Comparable data dependent on antibiotic concentration were obtained with each of the six antimicrobial agents investigated in this study. Because gonococci of Ti and T4 colony types have different rates of growth (11), the possibility existed that susceptibility to these antimicrobial agents may have been a function of cell growth rather than of colony type. To eliminate this possibility, Ti cultures and T4 cultures were grown to similar cell densities (3.4 x 107 CFU/ml) before the addition of ampicillin or penicillin. The data obtained (Table 2) showed that, although the T4 culture grew at a faster rate than the Ti culture, both virulent and avirulent gonococci had antibiotic susceptibilities similar to the data presented in the previous studies. Growth of elinical strains in the presence of antimicrobial agents. Bacteria of Ti and T4 colony types from several clinical strains were grown in the presence of the six antimicrobial agents. These strains had patterns of sus-
ceptibilities
to
chloramphenicol, erythromycin,
spectinomycin, and tetracycline similar to the patterns observed with strain F62. However, dif-
ferences in susceptibilities of Ti cultures and T4 cultures of these clinical strains to ampicillin and penicillin occurred only at higher concentrations of these antimicrobial agents. As shown in Table 3, Ti cultures of clinical strain USF2, a male urethral isolate, were more resistant than T4 cultures to the effects of ampicillin and penicillin at concentrations of 5.0 and 0.25 ug/ml, respectively, than at the lower concentrations of
) and T4 cultures (-----) in the presence of different FIG. 1. Growth of N. gonorrhoeae F62 Tl cultures ( antimicrobial agents. Deviations from the mean value obtained in three separate experiments are shown for each concentration point.
AMPICILLIN
P
100 80 60
-
40
-
20
-
0.001 100, 80 7
0.1 0.1
0.01
CHLORAMPHENICOL
1.0
10.0
TETRACYCLINE
IN.
60 _ 40 -1
20-i
4
0.1
5
1.0
1.0
10.0 0.1
10.0
ERYTHROMYCIN
100.
80. 60
40.
20.
0.01
0.1
1.0
10.0
100.0
SPECTINOMYCIN
100 80_
60_ 40_ 20-
1.0 CONCENTRATION (pg/mI)
47
100.0
48
ANTIMICROB. AGENTS CHEMOTHER.
LIM
TABLE 1. Growth of N. gonorrhoeae F62 in the presence of different antibiotiCsa Antibiotc
Concn
(ntlblotLc Xg/m1)
Ti
culture
CFU/ml (% of control cultureb)
T4 culture
1.22 x 109 1.12 X 109 8.17 X 104 (0.007 ± 0.005) 5.75 x 105 (0.052 ± 0.017) 0.50 3.42 x 105 (0.028 ± 0.021) 0.01 4.82 x 106 (0.430 ± 0.182) PeniciNlin 3.21 x 10" (26.349 ± 2.857) 0.40 1.49 x 10" (13.289 ± 1.163) Chloramphenicol 3.55 x 108 (29.121 ± 2.747) 0.10 8.48 X 107 (7.569 ± 0.413) Erythromycin 2.64 x 107 (2.166 ± 0.510) 8.85 x 106 (0.790 ± 0.264) 10.00 Spectinomycin 2.84 x 10" (23.267 ± 0.759) 0.30 9.69 x 107 (8.653 ± 2.367) Tetracychne a Bacteria of Ti and T4 colony types were grown in liquid MCDM at 36°C in a water bath shaker, as described in the text. After 6 h of incubation, samples were plated onto plates of GCB. The plates were incubated for 24 h at 36°C in 5% C02, and the CFU were counted. Samples from Ti cultures contained 90% Ti colony types and 10% T4 colony types, whereas samples from T4 cultures contained 100% T4 colony types. Data presented represent total CFU on the plates. Control cultures contained no antibiotics. b Values represent the mean and range of three separate experiments.
None AmpiciUlin
these antimicrobial agents used in studies with strain F62. The differences observed also were not as great as with the laboratory strain. DISCUSSION Since the introduction of penicillin as the antimicrobial agent of choice for the treatment of uncomplicated gonococcal infections, the resistance of N. gonorrhoeae to penicillin has increased steadily (13). In recent years gonococcal strains containing a plasmid-mediated beta-lactamase gene that degrades the beta-lactam ring of penicillin and makes the antibiotic ineffective in chemotherapy have been isolated in Great Britain (16), the Far East (15), and the United States (1). Other clinical isolates have been found to be partially or totally resistant to am-
picillin (28), spectinomycin (10), tetracycline (28), and other antimicrobial agents (3, 14, 23, 26). These antibiotic-resistant strains present a problem in management of gonorrhea and emphasize the need for further understanding of the mechanisms involved in antibiotic resistance.
Previous studies in this laboratory have shown that virulent (Ti colony type) and avirulent (T4 colony type) strains of N. gonorrhoeae have different rates of growth and metabolism (11). Other investigators have noted other differences in the two colonial types, such as morphology on solid medium (7, 9, 19), degree of piliation (8, 18), cell wall components (6, 17, 27, 29), and resistance to the bactericidal effects of serum (12). In this report we present evidence that virulent and avirulent strains of gonococci differ in their susceptibilities to antimicrobial agents. Our results show that bacteria of Ti and T4 colony types of N. gonorrhoeae have different degrees of susceptibility to antimicrobial agents. Ti cultures of laboratory strain F62 were more resistant than T4 cultures to the effects of ampicillin and penicillin, whereas T4 cultures were
more resistant to the effects of chloramphenicol, erythromycin, spectinomycin, and tetracycline. These differences in susceptibility could not be attributed to differences in growth rates of the two colony types, because similar results were obtained from cultures of both colony types grown for identical periods of time (Table 2). Both types of bacteria also were equally permeable to the uptake of crystal violet (data not shown). As demonstrated by Gustafsson et al. (4) and Guymon and Sparling (5), uptake of crystal violet is representative of the permeability of bacterial cells to antibiotics. Thus, decreases in susceptibility to antibiotics were not the result of decreased permeability to these
agents.
Susceptibility of Ti and T4 cultures to different antimicrobial agents, however, was dependent on the concentration of the particular agent used. Differences in susceptibilities of bacteria representing the two colony types to antimicrobial agents were greatest only at specific concentrations of the antibiotics investigated and negligible at other concentrations. This observation may explain why other investigators, using only single concentrations of antibiotics, have noted very little difference in susceptibilities of virulent and avirulent gonococci (7, 9). Clinical isolates of N. gonorrhoeae differed from laboratory strain F62 in their susceptibilities to ampicillin and penicillin. Differences in susceptibilities of clinical strains to ampicillin and penicillin occurred only at higher concentrations of the antibiotics. The extent of these differences also was greatly reduced as compared with strain F62. These observations indicated that the clinical strains, passaged only four to five times on artificial media, had developed an increased resistance to ampicillin and penicillin, as compared with strain F62, which has been passaged extensively (at least 2,500 times) on artificial media (9).
SUSCEPTIBILITY OF GONOCOCCI TO ANTIBIOTICS
VOL. 14, 1978
00
