Journal of Antimicrobial Chemotherapy (1978) 4, 39-45
A view of multiple drug resistance in Neisseria gonorrhoeae
Marda A. Chan and M. Goldner
Resistance to penicillin and tetracycline occurs in Neisseria gonorrhoeae yet the biochemical mechanism is unknown. Binding of f"C]-penicillin and absorption of [*H]-tetracycline were assayed in the range of minimum inhibitory concentration levels, to compare sensitive and multiple drug resistant strains of N. gonorrhoeae. Levels for both drugs were significantly higher in the sensitive strains. However, the effect of temperature differential on drug concentration pattern indicated that interference on the part of the resistant cell for penicillin related to passive diffusion and for tetracycline to facilitated diffusion. Thus, resistance in common to penicillin and tetracycline does not appear to rely on the outer membrane in N. gonorrhoeae. Introduction
The isolation of P-lactamase producing gonococci explains one mechanism of penicillin resistance. However, the majority of strains with decreased susceptibility to penicillin do not show the presence of P-lactamases. Reyn (1969) observed that gonococci that were resistant to penicillin often exhibited decreased sensitivity to other antibiotics which had been used in the treatment of gonorrhoea, namely tetracycline, erythromycin and streptomycin. Maier, BeUstein & Zubrzycki (1974) noticed this pattern of resistance particularly with penicillin and tetracycline, the most important drugs used in gonorrhoea therapy. The biochemical mechanism of resistance in multiple drug resistant (MDR) strains of Neisseria gonorrhoeae is unknown. In recent years, evidence has accumulated indicating that the cell wall complex of Gram negative organisms presents a diffusion barrier to antibiotics, preventing access to the target site (Leive, 1974). It therefore seemed feasible that the prevention of access of the antibiotics to the target sites of action could act as a common mechanism of resistance. Maness & Sparling (1973) have suggested that the multiple drug resistance displayed by the gonococci is due to an alteration in the outer membrane penetrability. It has been shown that the mechanism of both penicillin and tetracycline resistance may be associated with decreased binding and absorption of these drugs. Rodriguez & Saz (1975) have demonstrated decreased binding of [14C}-penicillin in resistant as compared to sensitive strains of gonococci. De Zeeuw (1968) and Franklin & Higginson (1970) have found that the mechanism of tetracycline resistance in bacteria seems to be associated with diminished absorption of the drug rather than a change in the affinity 39
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Department of Microbiology and Parasitology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A1
40
M. A. Chan and M. Goldner
for the ribosomal target site. Many investigators have demonstrated that tetracycline resistant bacteria accumulated less tetracycline than sensitive cells (Del Bene & Rogers, 1975; Franklin, 1967; Izaki, Kiuchi & Arima, 1966; Sompolinsky, Krawitz, Zaidenzaig & Abramova, 1970). The described experiments were designed to investigate the possible existence of a common mechanism of resistance, such as a diffusion barrier in the outer membrane, in N. gonorrhoeae. This approach is oriented to the direct comparison of sensitive and resistant strains as separate clinical isolates. Sensitive and MDR strains in both cases should then show a difference in binding and absorption of penicillin and tetracycline, respectively.
Bacterial strains The strains of N. gonorrhoeae used were clinical isolates obtained from the Women's College Hospital, Toronto and the Laboratory Services Branch, Ontario Ministry of Health, Toronto identified by morphological and biochemical characteristics (Reyn, 1974). Two resistant (WC 91 and PHL 160; M I C ^ l IU penicillin/ml, 1 ug tetracycline/ ml) and two sensitive (WC 12 and WC 5; MIC >005): (a)", at 37°C; (b), at 4°C.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Tetracycline absorption PHJ-Tetracydine was obtained as a crystalline solid from New England Nuclear Corporation, Dorval, Quebec. The specific activity of the antibiotic was 0-773 Ci/mmol. Resistant and sensitive gonococcal isolates were grown to mid exponential phase and 10-ml aliquots were prepared as with the labelled penicillin experiments. Concentrations of 0-25, 0-50, 0-75, 1-00 ug/ml ["HJ-tetracycline were each added to the 10-ml aliquots and the cells were then incubated for 20 min at 37°C and 4°C with shaking. Duplicate 3-ml samples were withdrawn, centrifuged at 30,000 xg at 4°C for 10 min and washed two times with equal volumes of modified Davis' minimal broth to remove extraneous tetracycline. The number of washings was limited to avoid any loss due to efflux (Franklin & Higginson, 1970). The cell pellets were resuspended in 3 ml modified Davis' minimal broth, filtered on Metricel membranes, dried and counted by the liquid scintillation technique.
M. A. Chan and M. Goldner
42
15
> Sensitive strains MDR strains
(o) 37*C
• Sensitive strains - » MDR strains
(b) 4*C
II ss Jo 0-7 e 0-3
175
275
075
1-75
275
(nmoles/ml)
Figure 2. Amount of [*H]-tetracycline absorbed with increasing concentration by two sensitive and two MDR strains of N. gonorrhoeae. Exponential phase cells (2x10* CFU/ml) were exposed to PHJtetracycline for 20 min. Each point represents the mean of 4 experimental trials for each sensitive and MDR strain, (a) At 37°C. Linear regression analysis showed that there was no departure from linearity for both, sensitive (P>0-25) and resistant (i'>0-50) cells. Using Student's t distribution, it was found that the slopes and the y-intercepts were significantly different (P 0-005). Using Student's / distribution, it was found that the slopes (.P>0-50) and the y-intercepts (i>>0-20) were not significantly different.
concentration. At very low penicillin concentrations, the sensitive and resistant strains appeared to bind the drug at about the same level. [*H]-Tetracycline absorbed
Figure 2(a) demonstrates the absorption of PHJ-tetracycline by the N. gonorrhoeae at a concentration range of MIC levels. It can be seen that the sensitive strains absorbed considerably more tetracycline at 37°C than the MDR strains, and in both cases the amount absorbed increased with increasing concentration. Figure 2(b) depicts the absorption of the drug at 4°C in the same concentration range. Both types of cells absorbed the same amount of drug at this temperature. Comparison of penicillin binding and tetracycline absorption
Figure 3(a) and (b) take note of the effect of the temperature differential when comparing tetracycline absorption with penicillin binding. A dramatic difference is observed in the concentration patterns. This difference in sensitive and resistant strains as seen in the histograms between tetracycline and penicillin relates to facilitated (temperature dependent) and passive (temperature independent) entry of the drugs into the organism. Discussion A difference was shown between sensitive and resistant strains of N. gonorrhoeae for both penicillin binding and tetracycline absorption. Penicillin sensitive gonococci bound significantly more penicillin than MDR strains at 37°C and 4°C [Figure l(a) and (b)]. Similarly, the sensitive strains absorbed significantly more tetracycline at 37°C
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
O75
Drug resistance in N. gonorrhoea*
(o)
• Passive diffusion S Sensltlv* strains R MDR strains E
1-5
• • S R
43
Facilitated diffusion Passive diffusion Sensitive strains MDR strains S
(b)
b 0-60
04O
07 / , "o
0-20
C-3
o
0-25 l4
C-75
0-75
1-25
[ C]-Penlclllin G (nmoles/ml)
1-75
275
5
[ H]-T»trocycllne (nmoles/ml)
Figure 3. Antibiotic transport in sensitive and MDR strains of N. gonorrhoeae. (a) ["CJ-penicillin transport. MDR strains bound less benzyl P*C>penicJUin than sensitive strains. The mechanism of penicillin resistance in the MDR strains relates to passive diffusion, (b) [*H]-tetracycline transport. MDR strains absorbed considerably less PHJ-tetracycline than sensitive strains. The mechanism of tetracycline resistance relates to facilitated diffusion.
than the MDR strains; however, they absorbed the same amount of drug at 4°C [Figure 2(a) and (b)]. A diffusion problem may exist in the MDR strains. Since the target sites for penicillin are located on the cytoplasmic membrane, the drug needs only to passively diffuse [Figure 3(a)] through the cell's envelope to reach its target sites (Franklin, 1973). The rate of diffusion in this case would be proportional to the external concentration of the drug. With the MDR gonococcal strains, the rate of diffusion of penicillin may be altered by some change or modification of the cell's envelope which prevents the drug from reaching its target site in a given time (Sarubbi, Sparling, Blackman & Lewis, 1975). Suginaka, Ichikawa & Kotani (1975) have supported this theory of a diffusion barrier in their work with Pseudomonas aeruginosa, which indicated that in the resistant organism penicillin was unable to effectively reach its target site as compared to EDTA treated cells exposed to benzyl [14C]-penicillin. However, since the pattern of penicillin binding did not clearly follow that described with other Gram-negative organisms (Strominger, Blumberg & Suginaka, 1971; Suginaka, Blumberg & Strominger, 1972), there may be a unique diffusion problem manifested in N. gonorrhoeae. Resistance may also be due in part to the nature of the binding sites. Since the sensitive strains seem to bind more penicillin than the resistant strains, it may be possible that the specific sites in the sensitive cells have a greater affinity for the drug than those sites in the resistant cells. Not only would this enhanced affinity result in greater binding of the drug by the sensitive cells but it would also lead to a greater susceptibility of the organisms to the drug. However, resistance may be dependent upon the ability of the cells to bind penicillin in terms of the number of sites, that is, with respect to the availability of the specific or target sites. Under these circumstances at the range of MIC levels, the difference in binding between penicillin sensitive and resistant strains would be significant but not extensive.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
^
44
M. A. Chan and M. GoJdner
Acknowledgements The authors are grateful to the J. P. Bickell Foundation of Toronto and Medical Research Council of Canada for their financial support. We are indebted to Dr Paul Corey for his help in the statistical analyses. References Del Bene, V. E. & Rogers, M. Comparison of tetracycline and minocycline transport in Escherichia coli. Antimicrobial Agents and Chemotherapy 7: 801-6 (1975). De Zeeuw, J. R. Accumulation of tetracyclines by Escherichia coli. Journal of Bacteriology 95: 498-506 (1968). Franklin, T. J. Resistance of E. coli to tetracycline: changes in permeability to tetracyclines in E. coli bearing transferable resistance factors. Biochemical Journal 105: 371-8 (1967). Franklin, T. J. Antibiotic transport in bacteria. In CRC Critical Reviews in Microbiology (Laskin, A. I. & Lechevalier, H., Eds). CRC Press, The Chemical Rubber Co., Cleveland (1973), pp. 253-72. Franklin, T. J. & Higginson, B. Active accumulation of tetracycline by E. coli. Biochemical Journal 116: 287-97 (1970). Gerhardt, P. & Heden, C. G. Concentrated cultures of gonococci in clear liquid medium. Proceedings of the Society for Experimental Biology & Medicine 105: 49-51 (1960).
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Unlike penicillin, tetracycline must pass through not only the outer membrane and peptidoglycan layers of the cell wall, but also through the cytoplasmic membrane in order to reach its target site, the 30S ribosomal subunit. The absorption of tetracycline by the sensitive strains based on the temperature differential continued to increase as the tetracycline concentration increased, whereas this absorption for the MDR strains did not increase over a basal level. Thus, processes related to the mechanism of resistance for tetracycline may be temperature dependent and similar to that described for other Gram-negative bacteria, that is, an exclusion of the drug by blocking its passage through the cytoplasmic membrane (Shipley & Olsen, 1974). If the level of tetracycline absorbed at 4°C by the MDR strains constituted essentially its passive diffusion through the outer membrane, then the small but gradual increase in tetracycline absorbed by the same strains at 37°C would reflect its low level of facilitated diffusion through the cytoplasmic membrane [Figure 3(b)]. Thus, the mechanism of tetracycline resistance may involve some change or modification of the cytoplasmic membrane that could affect facilitated diffusion of the drug. It is possible that a cytoplasmic membrane bound protein (Levy & McMurry 1974; Wojdani, Avtalion & Sompolinsky, 1976) exists and is associated with this change or modification in the MDR gonococcal strains. Although no difference has been found in ribosomal binding between sensitive and resistant Escherichia coli cells (De Zeeuw, 1968), the possibility of a change in ribosomal binding affinity in N. gonorrhoeae cannot be completely excluded. An obvious common mechanism of resistance such as an outer membrane diffusion barrier does not appear to operate in MDR strains of N. gonorrhoeae. A diffusion problem may exist for penicillin and tetracycline resistance but the type of diffusion problem seems different for each drug. It appears that the interference on the part of the resistant cell for penicillin relates to passive diffusion and for tetracycline to facilitated diffusion.
Drug resistance in N. gonorrhoeae
45
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Herbert, D., Phipps, P. J. & Strange, R. E. Chemical analysis of microbial cells. In Methods in Microbiology. Vol. 5B (Norris, J. R. & Ribbons, D. W., Eds). Academic Press, Inc., New York and London (1971), pp. 209-345. Izaki, K., Kiuchi, K. & Arima, K. Specificity and mechanisms of tetracycline resistance in a multiple drug-resistant strain of Escherichia coli. Journal of Bacteriology 91: 628-33 (1966). Lederberg, J. Isolation and characterization of biochemical mutants of bacteria. Methods in Medical Research 3: 5-22 (1950). Leive, L. The barrier function of the Gram-negative cell envelope. Annals of the New York Academy of Sciences 235: 109-27 (1974). Levy, S. E. & McMurry, L. Detection of an inducible membrane protein associated with Rfactor-mediated tetracycline resistance. Biochemical & Biophysical Research Communications 56: 1060-8 (1974). Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-75 (1951). Maier, T. W., Beilstein, H. R. & Zubrzycki, L. Multiple antibiotic resistance in Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 6: 22-8 (1974). Maness, M. J. & Sparling, P. F. Mulitple antibiotic resistance due to a single mutation in Neisseria gonorrhoeae. Journal of Infectious Diseases 128: 321-30 (1973). Reyn, A. Antibiotic sensitivity of gonococcal strains isolated in South-East Asia and Western Pacific regions in 1961-68. Bulletin of the World Health Organization 40: 257-62 (1969). Reyn, A. Gram-negative cocci and coccobacilli. In Sergey's Manual of Determinative Bacteriology. 8th Edition (Buchanan, R. E. & Gibbons, N. E., Eds). Williams and Wilkins Co., Baltimore (1974), pp. 427-9. Rodriguez, W. & Saz, A. K. Possible mechanism of decreased susceptibility of Neisseria gonorrhoeae to penicillin. Antimicrobial Agents and Chemotherapy 7: 788-92 (1975). Sarubbi, Jr., F. A., Sparling, P. F., Blackman, E. & Lewis, E. Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations. Journal of Bacteriology 124: 750-6 (1975). Shipley, P. L. & Olsen, R. H. Characteristics and expression of tetracycline resistance in Gramnegative bacteria carrying the Pseudomonas R-factor RP1. Antimicrobial Agents and Chemotherapy 6: 183-90 (1974). Sompolinsky, D., Krawitz, T., Zaidenzaig, Y. & Abramova, N. Inducible resistance to tetracycline in Staphylococcus aureus. Journal of General Microbiology 62: 341-9 (1970). Sparling, P. F., Sarubbi, Jr., F. A. & Blackman, E. Inheritance of low-level resistance to penicillin, tetracycline and chloramphenicol in Neisseria gonorrhoeae. Journal ofBacteriology V2A: 740-9 (1975). Strominger, J. L., Blumberg, P. M., Suginaka, H., Umbreit, J. & Wickus, G. G. How penicillin kills bacteria: progress and problems. Proceedings of the Royal Society of London. Series B 179: 369-83 (1971). Suginaka, H., Blumberg, P. M. & Strominger, J. L. Multiple penicillin binding components in Bacillus subtilis, Bacillus cereus, Staphylococcus aureus and Escherichia coli. Journal of Biological Chemistry 247: 5279-88 (1972). Suginaka, H., Ichikawa, A. & Kotani, S. Penicillin-resistant mechanisms in Pseudomonas aeruginosa: binding of penicillin to Pseudomonas aeruginosa KM 338. Antimicrobial Agents and Chemotherapy 7: 629-35 (1975). Wojdani, A., Avtalion, R. R. & Sompolinsky, D. Isolation and characterization of tetracycline resistance proteins from Staphylococcus aureus and Escherichia coli. Antimicrobial Agents and Chemotherapy 9: 526-34 (1976). {Manuscript accepted 30 June 1977)
A view of multiple drug resistance in Neisseria gonorrhoeae
Marda A. Chan and M. Goldner
Resistance to penicillin and tetracycline occurs in Neisseria gonorrhoeae yet the biochemical mechanism is unknown. Binding of f"C]-penicillin and absorption of [*H]-tetracycline were assayed in the range of minimum inhibitory concentration levels, to compare sensitive and multiple drug resistant strains of N. gonorrhoeae. Levels for both drugs were significantly higher in the sensitive strains. However, the effect of temperature differential on drug concentration pattern indicated that interference on the part of the resistant cell for penicillin related to passive diffusion and for tetracycline to facilitated diffusion. Thus, resistance in common to penicillin and tetracycline does not appear to rely on the outer membrane in N. gonorrhoeae. Introduction
The isolation of P-lactamase producing gonococci explains one mechanism of penicillin resistance. However, the majority of strains with decreased susceptibility to penicillin do not show the presence of P-lactamases. Reyn (1969) observed that gonococci that were resistant to penicillin often exhibited decreased sensitivity to other antibiotics which had been used in the treatment of gonorrhoea, namely tetracycline, erythromycin and streptomycin. Maier, BeUstein & Zubrzycki (1974) noticed this pattern of resistance particularly with penicillin and tetracycline, the most important drugs used in gonorrhoea therapy. The biochemical mechanism of resistance in multiple drug resistant (MDR) strains of Neisseria gonorrhoeae is unknown. In recent years, evidence has accumulated indicating that the cell wall complex of Gram negative organisms presents a diffusion barrier to antibiotics, preventing access to the target site (Leive, 1974). It therefore seemed feasible that the prevention of access of the antibiotics to the target sites of action could act as a common mechanism of resistance. Maness & Sparling (1973) have suggested that the multiple drug resistance displayed by the gonococci is due to an alteration in the outer membrane penetrability. It has been shown that the mechanism of both penicillin and tetracycline resistance may be associated with decreased binding and absorption of these drugs. Rodriguez & Saz (1975) have demonstrated decreased binding of [14C}-penicillin in resistant as compared to sensitive strains of gonococci. De Zeeuw (1968) and Franklin & Higginson (1970) have found that the mechanism of tetracycline resistance in bacteria seems to be associated with diminished absorption of the drug rather than a change in the affinity 39
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Department of Microbiology and Parasitology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A1
40
M. A. Chan and M. Goldner
for the ribosomal target site. Many investigators have demonstrated that tetracycline resistant bacteria accumulated less tetracycline than sensitive cells (Del Bene & Rogers, 1975; Franklin, 1967; Izaki, Kiuchi & Arima, 1966; Sompolinsky, Krawitz, Zaidenzaig & Abramova, 1970). The described experiments were designed to investigate the possible existence of a common mechanism of resistance, such as a diffusion barrier in the outer membrane, in N. gonorrhoeae. This approach is oriented to the direct comparison of sensitive and resistant strains as separate clinical isolates. Sensitive and MDR strains in both cases should then show a difference in binding and absorption of penicillin and tetracycline, respectively.
Bacterial strains The strains of N. gonorrhoeae used were clinical isolates obtained from the Women's College Hospital, Toronto and the Laboratory Services Branch, Ontario Ministry of Health, Toronto identified by morphological and biochemical characteristics (Reyn, 1974). Two resistant (WC 91 and PHL 160; M I C ^ l IU penicillin/ml, 1 ug tetracycline/ ml) and two sensitive (WC 12 and WC 5; MIC >005): (a)", at 37°C; (b), at 4°C.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Tetracycline absorption PHJ-Tetracydine was obtained as a crystalline solid from New England Nuclear Corporation, Dorval, Quebec. The specific activity of the antibiotic was 0-773 Ci/mmol. Resistant and sensitive gonococcal isolates were grown to mid exponential phase and 10-ml aliquots were prepared as with the labelled penicillin experiments. Concentrations of 0-25, 0-50, 0-75, 1-00 ug/ml ["HJ-tetracycline were each added to the 10-ml aliquots and the cells were then incubated for 20 min at 37°C and 4°C with shaking. Duplicate 3-ml samples were withdrawn, centrifuged at 30,000 xg at 4°C for 10 min and washed two times with equal volumes of modified Davis' minimal broth to remove extraneous tetracycline. The number of washings was limited to avoid any loss due to efflux (Franklin & Higginson, 1970). The cell pellets were resuspended in 3 ml modified Davis' minimal broth, filtered on Metricel membranes, dried and counted by the liquid scintillation technique.
M. A. Chan and M. Goldner
42
15
> Sensitive strains MDR strains
(o) 37*C
• Sensitive strains - » MDR strains
(b) 4*C
II ss Jo 0-7 e 0-3
175
275
075
1-75
275
(nmoles/ml)
Figure 2. Amount of [*H]-tetracycline absorbed with increasing concentration by two sensitive and two MDR strains of N. gonorrhoeae. Exponential phase cells (2x10* CFU/ml) were exposed to PHJtetracycline for 20 min. Each point represents the mean of 4 experimental trials for each sensitive and MDR strain, (a) At 37°C. Linear regression analysis showed that there was no departure from linearity for both, sensitive (P>0-25) and resistant (i'>0-50) cells. Using Student's t distribution, it was found that the slopes and the y-intercepts were significantly different (P 0-005). Using Student's / distribution, it was found that the slopes (.P>0-50) and the y-intercepts (i>>0-20) were not significantly different.
concentration. At very low penicillin concentrations, the sensitive and resistant strains appeared to bind the drug at about the same level. [*H]-Tetracycline absorbed
Figure 2(a) demonstrates the absorption of PHJ-tetracycline by the N. gonorrhoeae at a concentration range of MIC levels. It can be seen that the sensitive strains absorbed considerably more tetracycline at 37°C than the MDR strains, and in both cases the amount absorbed increased with increasing concentration. Figure 2(b) depicts the absorption of the drug at 4°C in the same concentration range. Both types of cells absorbed the same amount of drug at this temperature. Comparison of penicillin binding and tetracycline absorption
Figure 3(a) and (b) take note of the effect of the temperature differential when comparing tetracycline absorption with penicillin binding. A dramatic difference is observed in the concentration patterns. This difference in sensitive and resistant strains as seen in the histograms between tetracycline and penicillin relates to facilitated (temperature dependent) and passive (temperature independent) entry of the drugs into the organism. Discussion A difference was shown between sensitive and resistant strains of N. gonorrhoeae for both penicillin binding and tetracycline absorption. Penicillin sensitive gonococci bound significantly more penicillin than MDR strains at 37°C and 4°C [Figure l(a) and (b)]. Similarly, the sensitive strains absorbed significantly more tetracycline at 37°C
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
O75
Drug resistance in N. gonorrhoea*
(o)
• Passive diffusion S Sensltlv* strains R MDR strains E
1-5
• • S R
43
Facilitated diffusion Passive diffusion Sensitive strains MDR strains S
(b)
b 0-60
04O
07 / , "o
0-20
C-3
o
0-25 l4
C-75
0-75
1-25
[ C]-Penlclllin G (nmoles/ml)
1-75
275
5
[ H]-T»trocycllne (nmoles/ml)
Figure 3. Antibiotic transport in sensitive and MDR strains of N. gonorrhoeae. (a) ["CJ-penicillin transport. MDR strains bound less benzyl P*C>penicJUin than sensitive strains. The mechanism of penicillin resistance in the MDR strains relates to passive diffusion, (b) [*H]-tetracycline transport. MDR strains absorbed considerably less PHJ-tetracycline than sensitive strains. The mechanism of tetracycline resistance relates to facilitated diffusion.
than the MDR strains; however, they absorbed the same amount of drug at 4°C [Figure 2(a) and (b)]. A diffusion problem may exist in the MDR strains. Since the target sites for penicillin are located on the cytoplasmic membrane, the drug needs only to passively diffuse [Figure 3(a)] through the cell's envelope to reach its target sites (Franklin, 1973). The rate of diffusion in this case would be proportional to the external concentration of the drug. With the MDR gonococcal strains, the rate of diffusion of penicillin may be altered by some change or modification of the cell's envelope which prevents the drug from reaching its target site in a given time (Sarubbi, Sparling, Blackman & Lewis, 1975). Suginaka, Ichikawa & Kotani (1975) have supported this theory of a diffusion barrier in their work with Pseudomonas aeruginosa, which indicated that in the resistant organism penicillin was unable to effectively reach its target site as compared to EDTA treated cells exposed to benzyl [14C]-penicillin. However, since the pattern of penicillin binding did not clearly follow that described with other Gram-negative organisms (Strominger, Blumberg & Suginaka, 1971; Suginaka, Blumberg & Strominger, 1972), there may be a unique diffusion problem manifested in N. gonorrhoeae. Resistance may also be due in part to the nature of the binding sites. Since the sensitive strains seem to bind more penicillin than the resistant strains, it may be possible that the specific sites in the sensitive cells have a greater affinity for the drug than those sites in the resistant cells. Not only would this enhanced affinity result in greater binding of the drug by the sensitive cells but it would also lead to a greater susceptibility of the organisms to the drug. However, resistance may be dependent upon the ability of the cells to bind penicillin in terms of the number of sites, that is, with respect to the availability of the specific or target sites. Under these circumstances at the range of MIC levels, the difference in binding between penicillin sensitive and resistant strains would be significant but not extensive.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
^
44
M. A. Chan and M. GoJdner
Acknowledgements The authors are grateful to the J. P. Bickell Foundation of Toronto and Medical Research Council of Canada for their financial support. We are indebted to Dr Paul Corey for his help in the statistical analyses. References Del Bene, V. E. & Rogers, M. Comparison of tetracycline and minocycline transport in Escherichia coli. Antimicrobial Agents and Chemotherapy 7: 801-6 (1975). De Zeeuw, J. R. Accumulation of tetracyclines by Escherichia coli. Journal of Bacteriology 95: 498-506 (1968). Franklin, T. J. Resistance of E. coli to tetracycline: changes in permeability to tetracyclines in E. coli bearing transferable resistance factors. Biochemical Journal 105: 371-8 (1967). Franklin, T. J. Antibiotic transport in bacteria. In CRC Critical Reviews in Microbiology (Laskin, A. I. & Lechevalier, H., Eds). CRC Press, The Chemical Rubber Co., Cleveland (1973), pp. 253-72. Franklin, T. J. & Higginson, B. Active accumulation of tetracycline by E. coli. Biochemical Journal 116: 287-97 (1970). Gerhardt, P. & Heden, C. G. Concentrated cultures of gonococci in clear liquid medium. Proceedings of the Society for Experimental Biology & Medicine 105: 49-51 (1960).
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Unlike penicillin, tetracycline must pass through not only the outer membrane and peptidoglycan layers of the cell wall, but also through the cytoplasmic membrane in order to reach its target site, the 30S ribosomal subunit. The absorption of tetracycline by the sensitive strains based on the temperature differential continued to increase as the tetracycline concentration increased, whereas this absorption for the MDR strains did not increase over a basal level. Thus, processes related to the mechanism of resistance for tetracycline may be temperature dependent and similar to that described for other Gram-negative bacteria, that is, an exclusion of the drug by blocking its passage through the cytoplasmic membrane (Shipley & Olsen, 1974). If the level of tetracycline absorbed at 4°C by the MDR strains constituted essentially its passive diffusion through the outer membrane, then the small but gradual increase in tetracycline absorbed by the same strains at 37°C would reflect its low level of facilitated diffusion through the cytoplasmic membrane [Figure 3(b)]. Thus, the mechanism of tetracycline resistance may involve some change or modification of the cytoplasmic membrane that could affect facilitated diffusion of the drug. It is possible that a cytoplasmic membrane bound protein (Levy & McMurry 1974; Wojdani, Avtalion & Sompolinsky, 1976) exists and is associated with this change or modification in the MDR gonococcal strains. Although no difference has been found in ribosomal binding between sensitive and resistant Escherichia coli cells (De Zeeuw, 1968), the possibility of a change in ribosomal binding affinity in N. gonorrhoeae cannot be completely excluded. An obvious common mechanism of resistance such as an outer membrane diffusion barrier does not appear to operate in MDR strains of N. gonorrhoeae. A diffusion problem may exist for penicillin and tetracycline resistance but the type of diffusion problem seems different for each drug. It appears that the interference on the part of the resistant cell for penicillin relates to passive diffusion and for tetracycline to facilitated diffusion.
Drug resistance in N. gonorrhoeae
45
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on May 18, 2015
Herbert, D., Phipps, P. J. & Strange, R. E. Chemical analysis of microbial cells. In Methods in Microbiology. Vol. 5B (Norris, J. R. & Ribbons, D. W., Eds). Academic Press, Inc., New York and London (1971), pp. 209-345. Izaki, K., Kiuchi, K. & Arima, K. Specificity and mechanisms of tetracycline resistance in a multiple drug-resistant strain of Escherichia coli. Journal of Bacteriology 91: 628-33 (1966). Lederberg, J. Isolation and characterization of biochemical mutants of bacteria. Methods in Medical Research 3: 5-22 (1950). Leive, L. The barrier function of the Gram-negative cell envelope. Annals of the New York Academy of Sciences 235: 109-27 (1974). Levy, S. E. & McMurry, L. Detection of an inducible membrane protein associated with Rfactor-mediated tetracycline resistance. Biochemical & Biophysical Research Communications 56: 1060-8 (1974). Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-75 (1951). Maier, T. W., Beilstein, H. R. & Zubrzycki, L. Multiple antibiotic resistance in Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 6: 22-8 (1974). Maness, M. J. & Sparling, P. F. Mulitple antibiotic resistance due to a single mutation in Neisseria gonorrhoeae. Journal of Infectious Diseases 128: 321-30 (1973). Reyn, A. Antibiotic sensitivity of gonococcal strains isolated in South-East Asia and Western Pacific regions in 1961-68. Bulletin of the World Health Organization 40: 257-62 (1969). Reyn, A. Gram-negative cocci and coccobacilli. In Sergey's Manual of Determinative Bacteriology. 8th Edition (Buchanan, R. E. & Gibbons, N. E., Eds). Williams and Wilkins Co., Baltimore (1974), pp. 427-9. Rodriguez, W. & Saz, A. K. Possible mechanism of decreased susceptibility of Neisseria gonorrhoeae to penicillin. Antimicrobial Agents and Chemotherapy 7: 788-92 (1975). Sarubbi, Jr., F. A., Sparling, P. F., Blackman, E. & Lewis, E. Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations. Journal of Bacteriology 124: 750-6 (1975). Shipley, P. L. & Olsen, R. H. Characteristics and expression of tetracycline resistance in Gramnegative bacteria carrying the Pseudomonas R-factor RP1. Antimicrobial Agents and Chemotherapy 6: 183-90 (1974). Sompolinsky, D., Krawitz, T., Zaidenzaig, Y. & Abramova, N. Inducible resistance to tetracycline in Staphylococcus aureus. Journal of General Microbiology 62: 341-9 (1970). Sparling, P. F., Sarubbi, Jr., F. A. & Blackman, E. Inheritance of low-level resistance to penicillin, tetracycline and chloramphenicol in Neisseria gonorrhoeae. Journal ofBacteriology V2A: 740-9 (1975). Strominger, J. L., Blumberg, P. M., Suginaka, H., Umbreit, J. & Wickus, G. G. How penicillin kills bacteria: progress and problems. Proceedings of the Royal Society of London. Series B 179: 369-83 (1971). Suginaka, H., Blumberg, P. M. & Strominger, J. L. Multiple penicillin binding components in Bacillus subtilis, Bacillus cereus, Staphylococcus aureus and Escherichia coli. Journal of Biological Chemistry 247: 5279-88 (1972). Suginaka, H., Ichikawa, A. & Kotani, S. Penicillin-resistant mechanisms in Pseudomonas aeruginosa: binding of penicillin to Pseudomonas aeruginosa KM 338. Antimicrobial Agents and Chemotherapy 7: 629-35 (1975). Wojdani, A., Avtalion, R. R. & Sompolinsky, D. Isolation and characterization of tetracycline resistance proteins from Staphylococcus aureus and Escherichia coli. Antimicrobial Agents and Chemotherapy 9: 526-34 (1976). {Manuscript accepted 30 June 1977)
