ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1976, Copyright © 1976 American Society for Microbiology
p.
417-420
Vol. 10, No. 3
Printed in U.S.A.
Bacteriocin Production by Neisseria gonorrhoeae WILLIAM D. LAWTON,* MARY ANN BELLINGER, MARION SCHLING, ANID HASSAN A. GAAFAR Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201
Received for publication 18 April 1976
Seventeen apparently unrelated isolates of Neisseria gonorrhoeae out of 2,123 tested produced a diffusible growth-inhibitory substance against other gonococci. The inhibitor was destroyed by trypsin, not blocked by bovine serum albumin, and not soluble in chloroform-methanol; each isolate was resistant to the inhibitor it produced. Thus, the substance differs from previously described gonococcal inhibitors, and since it fits the description of a bacteriocin we designated it gonocin. The use of gonocin for typing was complicated by the observation that susceptibility to gonocin appears to depend on the gonococcal colony type. Bacteriocins are antibacterial proteins that are produced by bacteria and act against others of the same or related species (8). Since typing schemes are limited for Neisseria gonorrhoeae and since bacteriocin typing has been used to classify various microorganisms (2, 8), we and others have investigated the possibility of bacteriocin production by N. gonorrhoeae. Flynn and McEntegart (3) reported that 75 of 100 gonococcal isolates tested produced bacteriocins, but Walstad et al. (9) were unable to confirm their observation. The latter authors found that N. gonorrhoeae produce inhibitory fatty acids and lysophosphatidylethanolamine but not true bacteriocins. Knapp et al. (7) also found a nonspecific inhibitor but no bacteriocins produced by N. gonorrhoeae. Kingsbury (6) reported on bacteriocin production in the closely related Neisseria meningitidis. This report details our observations on gonococcal bacteriocin production.
MATERIALS AND METHODS Microorganisms and media. All of the isolates of N. gonorrhoeae used were received as patient specimens on Gonogrow transport medium (1) and were characterized as oxidase-positive, gram-negative diplococci that fermented glucose but not maltose. Cultures were stored at -70°C in GC broth containing IsoVitaleX (BBL) and 10% dimethyl sulfoxide. GC broth was made according to the formula for Difco GC base, except the agar was omitted. Although Difco GC base plus IsoVitaleX could be used for gonocin typing, clearer results were obtained with GC broth plus IsoVitaleX and 1% Difco purified agar. Gonocin assays. Isolates of N. gonorrhoeae to be tested for gonocin production were suspended in GC broth plus IsoVitaleX at approximately 108 cells/ml. A replicator with metal prongs was used to inoculate up.to 28 spots from the suspensions, each 5 mm in
diameter, on the surface of a 1% agar plate. After incubation for 1 day at 360C in a 5% C02 atmosphere, the spots were overlayered with 7 ml of soft agar (GC broth plus IsoVitaleX and 0.5% agar [Difco]) seeded with approximately 107 cells of an isolate to be tested for susceptibility to gonocin per ml. We found it unnecessary to treat the gonococcal spots with chloroform before pouring the soft-agar overlayer. The plates were reincubated overnight, and production of gonocin was measured as a clear zone extending approximately 5 to 8 mm from the edge of the gonocin-producing spot in the turbid lawn. Gonocin extracted from cells was assayed by pouring a seeded soft-agar layer on an uninoculated plate, allowing it to harden, and then placing a drop of the material to be tested on the surface and incubating the plate overnight at 36°C in a 5% C02 atmosphere. Inhibition was shown, as above, by a clear zone on the turbid lawn. Production of gonocin. Each gonocin-producing isolate of N. gonorrhoeae was inoculated into a 2liter flask containing a 300-ml base layer of Difco GC base with 2% agar (Difco) and a 200-ml top layer of GC broth plus IsoVitaleX. The flask was then shaken in a 5% C02 atmosphere on a rotary shaker at 100 rpm at 36°C for 24 h. The cells were sedimented by centrifugation at 16,000 x g for 10 min, and the supernatant was dialyzed overnight against distilled water and lyophilized. Trypsin and albumin treatment. Lyophilized gonocin was concentrated 10 times by resuspending at 50 mg/ml of 0.1 M sodium phosphate buffer, pH 6.2. For trypsin treatment, 0.5 ml of this suspension and 0.1 ml of Sigma type III trypsin (0.8 mg/ml) were incubated together at 360C for 3 h. To determine the effect of bovine serum albumin, the same procedure was used with 0.1 ml of 20% bovine serum albumin (Pentex) in place of trypsin.
RESULTS Initial screening. Each of 18 N. gonorrhoeae isolates was spotted on 18 agar plates and incubated overnight. Each isolate was then used in 417
418
LAWTON ET AL.
a soft-agar overlay on one plate of each set. Thus, each isolate was tested for both production of gonocin and susceptibility to gonocin with itself and 17 other isolates. This procedure was later repeated with another group of 72 isolates, for a total of 90 different gonococcal isolates. Only one isolate produced a large clear zone of inhibition in the indicator lawns of most of the other isolates tested (Fig. 1). This was isolate 16765, to which 62 (86%) of the isolates in the second group were susceptible. To determine if the inhibitor produced by 16765 is specific to N. gonorrhoeae, we tested it- with a variety of microorganisms. One of these, Moraxella osloensis 33413, showed a zone of inhibition larger than the zones seen in gonococcal indicator lawns. We therefore included M. osloensis 33413 as an indicator in our subsequent search for additional gonocin producers. Mass screening for gonocin producers. Two especially susceptible indicators, N. gonorrhoeae 8109 and M. osloensis 33413, were used to screen gonococcal isolates for their ability to produce gonocin. A total of 2,123 isolates of N. gonorrhoeae were tested over a period of a year, and 22 of these produced clear zones of inhibition with both indicators. A few of these 22 isolates were taken from the same patient a few
ANTIMIcROB. AGENTS CHEMOTHER.
weeks apart (treatment failures), and a few were from admitted sex contacts. When these duplications were omitted, we had 17 apparently unrelated gonococcal isolates that produced gonocin, or 0.8% of all the isolates tested. Differential production of gonocin by gonococci types 1 and 4. All of 14 single type 1 colonies (5) from isolate 16765 produced less gonocin than did 16 single type 4 colonies (5) from the same isolate. Three other gonocin producers were tested and behaved the same, but the demonstration of this was inconsistent, varying on different batches of medium. Type 1 cells shift rapidly to type 4 in vitro and, even when a single type 1 colony is used to make a spot of growth for testing gonocin production, the spot contains some type 4 cells. For this reason the finding remains uncertain. Type 1 cells may sometimes produce less gonocin than do type 4 cells; or, alternatively, type-1 cells may produce no gonocin, and the activity may result from the presence of a few type 4 cells in the type 1 colony. Large-scale production of crude gonocin. Isolate 16765, type 4, was used in an attempt at large-scale production of gonocin. Athough this isolate grew well in a biphasic GC broth medium (several 2,000-ml batches were produced), the supernatant did not contain enough gono-
FIG. 1. Inhibition zones surrounding two spots of N. gonorrhoeae 16765 in a lawn seeded with N. gonorrhoeae 903966.
VOL. 10, 1976
N. GONORRHOEAE BACTERIOCIN PRODUCTION
cin to assay. To achieve a 10-fold concentration, the supernatant was dialyzed overnight against distilled water, lyophilized, and resuspended at 50 mg/ml in distilled water. This material produced a clear zone of inhibition. An identical preparation from a non-gonocin-producing isolate showed no inhibitory zones in the indicator lawn. Attempts to obtain greater quantities of gonocin after rupturing the cells by freeze-thawing, sonication, or 5% chloroform treatment were unsuccessful. We also failed to obtain significant yields of gonocin after growing a confluent lawn of strain 16765 on GC agar and extracting the agar with water, pH 6.2 buffer, or chloroform-methanol or by testing the fluid expressed from such agar after a freeze-thaw cycle. Since the production of bacteriocins can often be increased by induction (6, 8), we made several attempts to increase the yield of gonocin by use of mitomycin C. Three different gonocinproducing isolates were tested, with mitomycin C concentrations of 0.1 to 10 ug/ml added at different stages of the growth curve, but no evidence was obtained to indicate a greater production of gonocin than was obtained from control cultures without mitomycin C.
419
Evidence for true gonocin. Since other workers (7, 9) have reported on inhibitory activity in N. gonorrhoeae but showed that the activity was not related to bacteriocins, we tested our crude gonocin extract from two isolates in several ways to demonstrate that it was different from the previously reported inhibitors (see Discussion). When 10-fold concentrated supernatants from gonocin-producing isolates 16765 and 82792 were treated with trypsin, the inhibitory activity disappeared, but treatment with 3% bovine serum albumin did not affect their activity (Fig. 2). Extraction of crude gonocin with chloroform-methanol (4) did not yield gonocin activity in the solvent. Removal of essentially all of the microorganisms from a spot of growth of isolate 16765 before adding an indicator lawn did not influence the zone of inhibition. Use of gonocin for typing. All 17 gonocinproducing isolates were investigated to determine whether susceptibility to gonocin could be used to type various N. gonorrhoeae isolates. Twelve of the gonocin producers appeared to give different typing patterns, and subsequent testing was concentrated on them. Our initial results with these 12 isolates were erratic, but when 15 isolates to be tested were
FIG. 2. Effect of trypsin and bovine serum aloumin (BSA) on activity of crude gonocin extract from N. gonorrhoeae 82792. The five spots on Difco GC base plus IsoVitaleX are: (A) trypsin plus buffer; (B) trypsin plus gonocin; (C) buffer plus gonocin; (D) BSA plus gonocin; (E) BSA plus buffer.
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LAWTON ET AL.
separated into Ti and T4 populations (5) reproducible patterns emerged. All 15 Ti isolates, but only 4 T4 isolates, were susceptible to all 12 gonocin producers. The remaining 11 T4 isolates were sensitive to one group of six producers and resistant to the other six. The resistance patterns were clearly reproducible, and our attempts to type other gonococcal isolates focused on T4 only. Typing with 60 T4 isolates indicated that at least two different gonocins were being produced and that the responses were as follows: (i) 36 test isolates were resistant to gonocin A and susceptible to gonocin B; (ii) 19 were susceptible to both gonocins; (iii) 1 was susceptible to gonocin A and resistant to gonocin B; and (iv) 4 were resistant to both gonocins. The gonocin A-producing strain was resistant to gonocin A but not to gonocin B; the gonocin B-producing strain was resistant to both gonocins. Both of the isolates used to demonstrate trypsin susceptibility produce gonocin A. As soon as our typing results indicated that at least two different inhibitors were produced, we used isolate 69992 to demonstrate that gonocin B was also destroyed by trypsin but unaffected by bovine serum albumin. DISCUSSION The search for bacteriocin activity in N. gonorrhoeae is complicated by the fact that all isolates appear to produce a substance with growth-inhibitory activity against other gonococci. This substance was shown by Walstad et al. (9) to be lysophosphatidylethanolamine and free fatty acids and is probably the same inhibitor subsequently described by Knapp et al. (7). We have observed this type of inhibitory activity with many of our gonococcal isolates, regardless of whether they produce gonocin. The inhibitor that we describe as gonocin differs from the previously described inhibitors in several ways. Most important, our inhibitor was destroyed by trypsin, indicating its protein nature. As expected with bacteriocins, no gonocin-producing strain was susceptible to its own gonocin when tested with a standard (50 mg/ ml) preparation of gonocin. Whereas the fatty acid inhibitor described by Walstad et al. (9) was completely blocked by addition of 2% bovine serum albumin, gonocin was unaffected. That inhibitor was also soluble in chloroformmethanol; gonocin was not. The inhibitor described by Knapp et al. (7) was inhibited by serum; gonocin was not. Its activity caused irregular inhibition zones from 1 to 3 mm beyond the spot and was dependent on the presence of growth of the producing strains. In contrast,
ANTIMICROB. AGENTS CHEMOTHER.
gonocin-producing isolates created zones extending 5 to 8 mm beyond the spot, and the zone of inhibition did not depend on the continued presence of the producing spot of growth. The "bacteriocin-like" inhibition described by Flynn and McEntegart (3) was not substantiated by Walstad et al. (9), and Knapp et al. (7) suggested that the non-bacteriocin inhibitor described by them accounted for the observation of Flynn and McEntegart. For these reasons, we believe that we have demonstrated true bacteriocin (gonocin) production inN. gonorrhoeae for the first time. The usefulness of gonocin for typing gonococcal isolates remains in question. Only 0.8% of the strains tested produced gonocin, so typing isolates by their ability to produce gonocin would not be worthwhile. Typing by determining susceptibility to gonocin may be developed into a useful technique, but it is complicated by the fact that the susceptibility of an isolate can depend on its colonial type. Even when 60 type 4 isolates were selected (to obtain maximum differentiation by resistance patterns to two gonocins), 92% of the strains fell into only two groups. However, the technique could become more useful if more than two different gonocins were identified. ACKNOWLEDGMENT We thank Isobel DeForge for technical assistance in screening cultures for gonocin production.
LITERATURE CITED 1. Chen, N. C., S. S. Hipp, W. D. Lawton, and H. A. Gaafar. 1974. Gonogrow, an improved, selective medium for the isolation of Neisseria gonorrhoeae. Health Lab. Sci. 11:173-177. 2. Counts, G. W., L. Seeley, and H. N. Beaty. 1971. Identification of an epidemic strain ofNeisseria meningitidis by bacteriocin typing. J. Infect. Dis. 124:26-32. 3. Flynn, J., and M. G. McEnteWt. 1972. Bacteriocins from Neisseria gonorrhoeae and their possible role in epidemiological studies. J. Clin. Pathol. 25:60-61. 4. Garbus, J., H. F. DeLuca, M. E. Loomans, and F. M. Strong. 1963. The rapid incorporation of phosphate into mitochondrial lipids. J. Biol. Chem. 238:59-63. 5. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. L. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274-1279. 6. Kingsbury, D. T. 1966. Bacteriocin production by strains of Neisseria meningitidis. J. Bacteriol.
91:1696-1699. 7. Knapp, J. S., S. Falkow, and K. K. Holmes. 1975. Reevaluation of bacteriocinogeny in Neisseria gonorrhoeae. J. Clin. Pathol. 28:274-278. 8. Reeves, P. 1965. The bacteriocins. Bacteriol. Rev. 29:2445. 9. Walstad, D. L., R. C. Reitz, and P. F. Sparling. 1974. Growth inhibition among strains of Neisseria gonorrhoeae due to production of inhibitory free fatty acids
and lysophosphatidylethanolamine: absence of bacteriocins. Infect. Immun. 10:481-488.
p.
417-420
Vol. 10, No. 3
Printed in U.S.A.
Bacteriocin Production by Neisseria gonorrhoeae WILLIAM D. LAWTON,* MARY ANN BELLINGER, MARION SCHLING, ANID HASSAN A. GAAFAR Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201
Received for publication 18 April 1976
Seventeen apparently unrelated isolates of Neisseria gonorrhoeae out of 2,123 tested produced a diffusible growth-inhibitory substance against other gonococci. The inhibitor was destroyed by trypsin, not blocked by bovine serum albumin, and not soluble in chloroform-methanol; each isolate was resistant to the inhibitor it produced. Thus, the substance differs from previously described gonococcal inhibitors, and since it fits the description of a bacteriocin we designated it gonocin. The use of gonocin for typing was complicated by the observation that susceptibility to gonocin appears to depend on the gonococcal colony type. Bacteriocins are antibacterial proteins that are produced by bacteria and act against others of the same or related species (8). Since typing schemes are limited for Neisseria gonorrhoeae and since bacteriocin typing has been used to classify various microorganisms (2, 8), we and others have investigated the possibility of bacteriocin production by N. gonorrhoeae. Flynn and McEntegart (3) reported that 75 of 100 gonococcal isolates tested produced bacteriocins, but Walstad et al. (9) were unable to confirm their observation. The latter authors found that N. gonorrhoeae produce inhibitory fatty acids and lysophosphatidylethanolamine but not true bacteriocins. Knapp et al. (7) also found a nonspecific inhibitor but no bacteriocins produced by N. gonorrhoeae. Kingsbury (6) reported on bacteriocin production in the closely related Neisseria meningitidis. This report details our observations on gonococcal bacteriocin production.
MATERIALS AND METHODS Microorganisms and media. All of the isolates of N. gonorrhoeae used were received as patient specimens on Gonogrow transport medium (1) and were characterized as oxidase-positive, gram-negative diplococci that fermented glucose but not maltose. Cultures were stored at -70°C in GC broth containing IsoVitaleX (BBL) and 10% dimethyl sulfoxide. GC broth was made according to the formula for Difco GC base, except the agar was omitted. Although Difco GC base plus IsoVitaleX could be used for gonocin typing, clearer results were obtained with GC broth plus IsoVitaleX and 1% Difco purified agar. Gonocin assays. Isolates of N. gonorrhoeae to be tested for gonocin production were suspended in GC broth plus IsoVitaleX at approximately 108 cells/ml. A replicator with metal prongs was used to inoculate up.to 28 spots from the suspensions, each 5 mm in
diameter, on the surface of a 1% agar plate. After incubation for 1 day at 360C in a 5% C02 atmosphere, the spots were overlayered with 7 ml of soft agar (GC broth plus IsoVitaleX and 0.5% agar [Difco]) seeded with approximately 107 cells of an isolate to be tested for susceptibility to gonocin per ml. We found it unnecessary to treat the gonococcal spots with chloroform before pouring the soft-agar overlayer. The plates were reincubated overnight, and production of gonocin was measured as a clear zone extending approximately 5 to 8 mm from the edge of the gonocin-producing spot in the turbid lawn. Gonocin extracted from cells was assayed by pouring a seeded soft-agar layer on an uninoculated plate, allowing it to harden, and then placing a drop of the material to be tested on the surface and incubating the plate overnight at 36°C in a 5% C02 atmosphere. Inhibition was shown, as above, by a clear zone on the turbid lawn. Production of gonocin. Each gonocin-producing isolate of N. gonorrhoeae was inoculated into a 2liter flask containing a 300-ml base layer of Difco GC base with 2% agar (Difco) and a 200-ml top layer of GC broth plus IsoVitaleX. The flask was then shaken in a 5% C02 atmosphere on a rotary shaker at 100 rpm at 36°C for 24 h. The cells were sedimented by centrifugation at 16,000 x g for 10 min, and the supernatant was dialyzed overnight against distilled water and lyophilized. Trypsin and albumin treatment. Lyophilized gonocin was concentrated 10 times by resuspending at 50 mg/ml of 0.1 M sodium phosphate buffer, pH 6.2. For trypsin treatment, 0.5 ml of this suspension and 0.1 ml of Sigma type III trypsin (0.8 mg/ml) were incubated together at 360C for 3 h. To determine the effect of bovine serum albumin, the same procedure was used with 0.1 ml of 20% bovine serum albumin (Pentex) in place of trypsin.
RESULTS Initial screening. Each of 18 N. gonorrhoeae isolates was spotted on 18 agar plates and incubated overnight. Each isolate was then used in 417
418
LAWTON ET AL.
a soft-agar overlay on one plate of each set. Thus, each isolate was tested for both production of gonocin and susceptibility to gonocin with itself and 17 other isolates. This procedure was later repeated with another group of 72 isolates, for a total of 90 different gonococcal isolates. Only one isolate produced a large clear zone of inhibition in the indicator lawns of most of the other isolates tested (Fig. 1). This was isolate 16765, to which 62 (86%) of the isolates in the second group were susceptible. To determine if the inhibitor produced by 16765 is specific to N. gonorrhoeae, we tested it- with a variety of microorganisms. One of these, Moraxella osloensis 33413, showed a zone of inhibition larger than the zones seen in gonococcal indicator lawns. We therefore included M. osloensis 33413 as an indicator in our subsequent search for additional gonocin producers. Mass screening for gonocin producers. Two especially susceptible indicators, N. gonorrhoeae 8109 and M. osloensis 33413, were used to screen gonococcal isolates for their ability to produce gonocin. A total of 2,123 isolates of N. gonorrhoeae were tested over a period of a year, and 22 of these produced clear zones of inhibition with both indicators. A few of these 22 isolates were taken from the same patient a few
ANTIMIcROB. AGENTS CHEMOTHER.
weeks apart (treatment failures), and a few were from admitted sex contacts. When these duplications were omitted, we had 17 apparently unrelated gonococcal isolates that produced gonocin, or 0.8% of all the isolates tested. Differential production of gonocin by gonococci types 1 and 4. All of 14 single type 1 colonies (5) from isolate 16765 produced less gonocin than did 16 single type 4 colonies (5) from the same isolate. Three other gonocin producers were tested and behaved the same, but the demonstration of this was inconsistent, varying on different batches of medium. Type 1 cells shift rapidly to type 4 in vitro and, even when a single type 1 colony is used to make a spot of growth for testing gonocin production, the spot contains some type 4 cells. For this reason the finding remains uncertain. Type 1 cells may sometimes produce less gonocin than do type 4 cells; or, alternatively, type-1 cells may produce no gonocin, and the activity may result from the presence of a few type 4 cells in the type 1 colony. Large-scale production of crude gonocin. Isolate 16765, type 4, was used in an attempt at large-scale production of gonocin. Athough this isolate grew well in a biphasic GC broth medium (several 2,000-ml batches were produced), the supernatant did not contain enough gono-
FIG. 1. Inhibition zones surrounding two spots of N. gonorrhoeae 16765 in a lawn seeded with N. gonorrhoeae 903966.
VOL. 10, 1976
N. GONORRHOEAE BACTERIOCIN PRODUCTION
cin to assay. To achieve a 10-fold concentration, the supernatant was dialyzed overnight against distilled water, lyophilized, and resuspended at 50 mg/ml in distilled water. This material produced a clear zone of inhibition. An identical preparation from a non-gonocin-producing isolate showed no inhibitory zones in the indicator lawn. Attempts to obtain greater quantities of gonocin after rupturing the cells by freeze-thawing, sonication, or 5% chloroform treatment were unsuccessful. We also failed to obtain significant yields of gonocin after growing a confluent lawn of strain 16765 on GC agar and extracting the agar with water, pH 6.2 buffer, or chloroform-methanol or by testing the fluid expressed from such agar after a freeze-thaw cycle. Since the production of bacteriocins can often be increased by induction (6, 8), we made several attempts to increase the yield of gonocin by use of mitomycin C. Three different gonocinproducing isolates were tested, with mitomycin C concentrations of 0.1 to 10 ug/ml added at different stages of the growth curve, but no evidence was obtained to indicate a greater production of gonocin than was obtained from control cultures without mitomycin C.
419
Evidence for true gonocin. Since other workers (7, 9) have reported on inhibitory activity in N. gonorrhoeae but showed that the activity was not related to bacteriocins, we tested our crude gonocin extract from two isolates in several ways to demonstrate that it was different from the previously reported inhibitors (see Discussion). When 10-fold concentrated supernatants from gonocin-producing isolates 16765 and 82792 were treated with trypsin, the inhibitory activity disappeared, but treatment with 3% bovine serum albumin did not affect their activity (Fig. 2). Extraction of crude gonocin with chloroform-methanol (4) did not yield gonocin activity in the solvent. Removal of essentially all of the microorganisms from a spot of growth of isolate 16765 before adding an indicator lawn did not influence the zone of inhibition. Use of gonocin for typing. All 17 gonocinproducing isolates were investigated to determine whether susceptibility to gonocin could be used to type various N. gonorrhoeae isolates. Twelve of the gonocin producers appeared to give different typing patterns, and subsequent testing was concentrated on them. Our initial results with these 12 isolates were erratic, but when 15 isolates to be tested were
FIG. 2. Effect of trypsin and bovine serum aloumin (BSA) on activity of crude gonocin extract from N. gonorrhoeae 82792. The five spots on Difco GC base plus IsoVitaleX are: (A) trypsin plus buffer; (B) trypsin plus gonocin; (C) buffer plus gonocin; (D) BSA plus gonocin; (E) BSA plus buffer.
420
LAWTON ET AL.
separated into Ti and T4 populations (5) reproducible patterns emerged. All 15 Ti isolates, but only 4 T4 isolates, were susceptible to all 12 gonocin producers. The remaining 11 T4 isolates were sensitive to one group of six producers and resistant to the other six. The resistance patterns were clearly reproducible, and our attempts to type other gonococcal isolates focused on T4 only. Typing with 60 T4 isolates indicated that at least two different gonocins were being produced and that the responses were as follows: (i) 36 test isolates were resistant to gonocin A and susceptible to gonocin B; (ii) 19 were susceptible to both gonocins; (iii) 1 was susceptible to gonocin A and resistant to gonocin B; and (iv) 4 were resistant to both gonocins. The gonocin A-producing strain was resistant to gonocin A but not to gonocin B; the gonocin B-producing strain was resistant to both gonocins. Both of the isolates used to demonstrate trypsin susceptibility produce gonocin A. As soon as our typing results indicated that at least two different inhibitors were produced, we used isolate 69992 to demonstrate that gonocin B was also destroyed by trypsin but unaffected by bovine serum albumin. DISCUSSION The search for bacteriocin activity in N. gonorrhoeae is complicated by the fact that all isolates appear to produce a substance with growth-inhibitory activity against other gonococci. This substance was shown by Walstad et al. (9) to be lysophosphatidylethanolamine and free fatty acids and is probably the same inhibitor subsequently described by Knapp et al. (7). We have observed this type of inhibitory activity with many of our gonococcal isolates, regardless of whether they produce gonocin. The inhibitor that we describe as gonocin differs from the previously described inhibitors in several ways. Most important, our inhibitor was destroyed by trypsin, indicating its protein nature. As expected with bacteriocins, no gonocin-producing strain was susceptible to its own gonocin when tested with a standard (50 mg/ ml) preparation of gonocin. Whereas the fatty acid inhibitor described by Walstad et al. (9) was completely blocked by addition of 2% bovine serum albumin, gonocin was unaffected. That inhibitor was also soluble in chloroformmethanol; gonocin was not. The inhibitor described by Knapp et al. (7) was inhibited by serum; gonocin was not. Its activity caused irregular inhibition zones from 1 to 3 mm beyond the spot and was dependent on the presence of growth of the producing strains. In contrast,
ANTIMICROB. AGENTS CHEMOTHER.
gonocin-producing isolates created zones extending 5 to 8 mm beyond the spot, and the zone of inhibition did not depend on the continued presence of the producing spot of growth. The "bacteriocin-like" inhibition described by Flynn and McEntegart (3) was not substantiated by Walstad et al. (9), and Knapp et al. (7) suggested that the non-bacteriocin inhibitor described by them accounted for the observation of Flynn and McEntegart. For these reasons, we believe that we have demonstrated true bacteriocin (gonocin) production inN. gonorrhoeae for the first time. The usefulness of gonocin for typing gonococcal isolates remains in question. Only 0.8% of the strains tested produced gonocin, so typing isolates by their ability to produce gonocin would not be worthwhile. Typing by determining susceptibility to gonocin may be developed into a useful technique, but it is complicated by the fact that the susceptibility of an isolate can depend on its colonial type. Even when 60 type 4 isolates were selected (to obtain maximum differentiation by resistance patterns to two gonocins), 92% of the strains fell into only two groups. However, the technique could become more useful if more than two different gonocins were identified. ACKNOWLEDGMENT We thank Isobel DeForge for technical assistance in screening cultures for gonocin production.
LITERATURE CITED 1. Chen, N. C., S. S. Hipp, W. D. Lawton, and H. A. Gaafar. 1974. Gonogrow, an improved, selective medium for the isolation of Neisseria gonorrhoeae. Health Lab. Sci. 11:173-177. 2. Counts, G. W., L. Seeley, and H. N. Beaty. 1971. Identification of an epidemic strain ofNeisseria meningitidis by bacteriocin typing. J. Infect. Dis. 124:26-32. 3. Flynn, J., and M. G. McEnteWt. 1972. Bacteriocins from Neisseria gonorrhoeae and their possible role in epidemiological studies. J. Clin. Pathol. 25:60-61. 4. Garbus, J., H. F. DeLuca, M. E. Loomans, and F. M. Strong. 1963. The rapid incorporation of phosphate into mitochondrial lipids. J. Biol. Chem. 238:59-63. 5. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. L. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274-1279. 6. Kingsbury, D. T. 1966. Bacteriocin production by strains of Neisseria meningitidis. J. Bacteriol.
91:1696-1699. 7. Knapp, J. S., S. Falkow, and K. K. Holmes. 1975. Reevaluation of bacteriocinogeny in Neisseria gonorrhoeae. J. Clin. Pathol. 28:274-278. 8. Reeves, P. 1965. The bacteriocins. Bacteriol. Rev. 29:2445. 9. Walstad, D. L., R. C. Reitz, and P. F. Sparling. 1974. Growth inhibition among strains of Neisseria gonorrhoeae due to production of inhibitory free fatty acids
and lysophosphatidylethanolamine: absence of bacteriocins. Infect. Immun. 10:481-488.
