Vol. 15, No. 2 Printed in U.S.A.
INFECTION AND IMMUNrIY, Feb. 1977, p. 663-664 Copyright X 1977 American Society for Microbiology
NOTES Production of a Capsule by Neisseria gonorrhoeae WALTER P. RICHARDSON' AND JERALD C. SADOFF* Department ofBacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012
Received for publication 28 July 1976
Neisseria gonorrhoeae can be induced to form a large capsule that can be visualized with India ink. Encapsulated gonococci appeared to be resistant to non-antibody-mediated phagocytosis compared with unencapsulated gonococci of the same strain and colony type.
The presence of a polysaccharide capsule on Neisseria gonorrhoeae (GC), comparable to that of the meningococcus, was suggested as early as 1937 by Wolfgang Casper (1). More recently, fluorescence data (2) suggested the presence of a capsule in freshly isolated strains. A capsulelike effect can be detected in fresh GC isolates by mixing viable GC with a solution of India ink and methylene blue and observing the bacteria by high-power light microscopy. By this technique we noted that capsule production was inconstant and rapidly lost on in vitro passage with a variety of solid and liquid media. During the course of this work, GC from a culture accidentally contaminated with microaerophilic "viridans streptococci" (SV) demonstrated the largest capsules we had seen. A technique was developed to make use of this phonemenon. Petri dishes containing an enriched chocolate agar (GC medium base, Difco Laboratories, Detroit, Mich.; 1% hemoglobin, Difco Laboratories; 1% IsoVitaleX, BBL, Baltimore, Md.) were first inoculated with 104 log-phase SV organisms. The agar was then inverted, inoculated side down, into a fresh petri dish, and approximately 106 log-phase Neisseria organisms (grown in medium 199, GIBCO, Grand Island, N.Y., containing 1% IsoVitaleX and 1% glucose) were spread on the uninoculated side. The plates were incubated in 10% CO2 at 370C for 16 to 24 h until the India ink-wet preparation indicated that the GC were encapsulated. Isolates of GC, as well as the other Neisseria species, N. meningitidis, N. subflava, and N. sicca, were tested for capsule production before and after growth with SV. The GC isolates that were passed many times in vitro had no capsule I Present address: Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.
or, at most, an equivocal capsule (Table 1). When these isolates were grown on the same media but in the presence of SV, definite largecapsule production similar to that shown in Fig. 1 was observed. The two fresh GC strains used had relatively small capsules when grown on the enriched agar without SV, but these capsules appeared considerably larger when the strain was grown in the presence of SV. It should be noted that the SV strain was the only bacterial strain we tested for capsule-inducing properties in this system. In the presence of SV, the thickness ofthe GC capsules was one-half to one times the diameter of the bacterial cell body and had somewhat fuzzy, irregular borders. These capsules could be washed off easily by forcing a suspension of the bacteria through an 18-gauge needle. The isolate of N. meningitidis produced visible capsules when cultured with or without the SV. The isolates of N. sicca and N. subflava produced small, sharply defined capsules when grown in the presence of SV; however, these capsules were distinct from those of GC in that they were not removable by gentle mechanical means. So called virulent colony types (Ti) and the less virulent colony type (T4) (3) derived from GC strains 134 and 230, respectively, were tested by this system; both colony types produced large capsules. Each of the colony types was monitored for any change, after production of the capsule, by passage onto colony typing medium (3). No change in colony type was observed. Having established conditions under which GC produced capsules, we investigated the functional role such a capsule might play. The peripheral human blood leukocyte slide phagocytosis test of Swanson et al. (4) was adopted to determine whether capsules provided resist-
663
664
NOTES
INFECT. IMMUN.
TABLE 1. Capsule production by Neisseria species a Neisseria isolate
N. gonorrhoeae 230 120 108 134 u17 u19
No. of previous in vitro passes
>20 >20 10 5 3 3
N. meningiditis 138I
Capsule production
Without SV
With SV
0 0
++ ++ ++ ++
+
± +
++
+
++
++
++
N. subflava Three strains
5
0
±
N. sicca Three strains
5
0
+
Organisms were grown with or without "viridans streptococci" and observed in India ink-methylene blue preparations as described in the text, and the capsules were scored as follows: 0, no capsule; ±, equivocal capsule; +, maximum capsule thickness one-half the diameter of the blue-stained organism; + +, capsule thickness greater than one-half the diameter of the organism. a
FIG. 1. GC grown in the presence of SV were smeared onto a glass slide, and 1 drop of 5% methylene blue in ethanol and 1 drop of India ink were quickly added and mixed on the slide. A cover slip was put over the preparation, and the bacteria were observed at a magnification of xl ,OOO. The bacterial bodies stain blue, and the capsules appear as unstained halos against the black India ink.
to non-antibody-mediated phagocytosis. Unencapsulated GC were attached to or ingested by polymorphonuclear leukocytes (PMNs) quite easily in this system. In one experiment with unencapsulated GC 108 (T4), 88% of 400 PMNs counted had one or more organisms attached or ingested, with the averance
TABLE 2. Phagocytosis of encapsulated versus unencapsulated N. gonorrhoeae Gonococcal strain
Encapsulated a (%)
Unencapsulated a (%)
104 31 91 19 88 108 24 93 125 20 78 134 24 76 230 u12 26 92 a Percentage of PMNs with one or more gonococcal organisms either attached or ingested. Equal numbers of encapsulated or unencapsulated organisms (between 107 and 108 colony-forming units, depending on the experiment) suspended in 1 ml of medium 199 containing 0.01% bovine serum albumin (Armour Pharmaceutical Co., Chicago, Ill.) were added simultaneously to -5 x 106 human PMNs (isolated from a single volunteer) attached to Lab Tek tissue culture chamber slides (Miles Laboratories, Naperville, Ill.), and incubated with shaking at 370C for 20 min. Slides were then washed, fixed with methanol, and stained with Giemsa for 90 min.
age being 7.8 organisms per PMN. In contrast, only 19% of 400 cells counted could be found with GC attached or ingested in the leukocyte wells simultaneously incubated with encapsulated GC 108 (T4) (average, 1.6 organisms per PMN). Table 2 shows the results of six sets of paired experiments demonstrating the resistance of encapsulated GC to attachment and ingestion by leukocytes. That the elaboration of a capsule by GC is associated with increased resistance to phagocytosis suggests a functional role for the capsule. Efforts to chemically and immunologically characterize the capsule are underway, so that its true significance can be determined. We thank Kathyrn K. Yamada for technical assistance.
LITERATURE CITED 1. Casper, W. A. 1937. The preparation of the type-specific carbohydrates of gonococi. J. Immunol. 23:421-439. 2. Deacon, W. E., W. L. Peacock, E. M. Freeman, and A. Harris. 1959. Identification of Neisseria gonorrhoeae by means of fluorescent antibodies. Proc. Soc. Exp. Biol. Med. 101:322-325. 3. Kellogg, D. S., Jr., I. R. Cohen, L. C. Norins, A. L. Schroeter, and G. Reising. 1968. Neisseria gonorrhoeae. II. Colonial variation and pathogenicity during 35 months in vitro. J. Bacteriol. 96:596-605. 4. Swanson, J., E. Sparks, B. Zeligs, M. A. Siam, and C. Parrot. 1976. Studies on gonococcus infection. V. Observations on in vitro interactions of gonococci and human neutrophils. J. Infect. Dis. 133:441-447.
INFECTION AND IMMUNrIY, Feb. 1977, p. 663-664 Copyright X 1977 American Society for Microbiology
NOTES Production of a Capsule by Neisseria gonorrhoeae WALTER P. RICHARDSON' AND JERALD C. SADOFF* Department ofBacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012
Received for publication 28 July 1976
Neisseria gonorrhoeae can be induced to form a large capsule that can be visualized with India ink. Encapsulated gonococci appeared to be resistant to non-antibody-mediated phagocytosis compared with unencapsulated gonococci of the same strain and colony type.
The presence of a polysaccharide capsule on Neisseria gonorrhoeae (GC), comparable to that of the meningococcus, was suggested as early as 1937 by Wolfgang Casper (1). More recently, fluorescence data (2) suggested the presence of a capsule in freshly isolated strains. A capsulelike effect can be detected in fresh GC isolates by mixing viable GC with a solution of India ink and methylene blue and observing the bacteria by high-power light microscopy. By this technique we noted that capsule production was inconstant and rapidly lost on in vitro passage with a variety of solid and liquid media. During the course of this work, GC from a culture accidentally contaminated with microaerophilic "viridans streptococci" (SV) demonstrated the largest capsules we had seen. A technique was developed to make use of this phonemenon. Petri dishes containing an enriched chocolate agar (GC medium base, Difco Laboratories, Detroit, Mich.; 1% hemoglobin, Difco Laboratories; 1% IsoVitaleX, BBL, Baltimore, Md.) were first inoculated with 104 log-phase SV organisms. The agar was then inverted, inoculated side down, into a fresh petri dish, and approximately 106 log-phase Neisseria organisms (grown in medium 199, GIBCO, Grand Island, N.Y., containing 1% IsoVitaleX and 1% glucose) were spread on the uninoculated side. The plates were incubated in 10% CO2 at 370C for 16 to 24 h until the India ink-wet preparation indicated that the GC were encapsulated. Isolates of GC, as well as the other Neisseria species, N. meningitidis, N. subflava, and N. sicca, were tested for capsule production before and after growth with SV. The GC isolates that were passed many times in vitro had no capsule I Present address: Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.
or, at most, an equivocal capsule (Table 1). When these isolates were grown on the same media but in the presence of SV, definite largecapsule production similar to that shown in Fig. 1 was observed. The two fresh GC strains used had relatively small capsules when grown on the enriched agar without SV, but these capsules appeared considerably larger when the strain was grown in the presence of SV. It should be noted that the SV strain was the only bacterial strain we tested for capsule-inducing properties in this system. In the presence of SV, the thickness ofthe GC capsules was one-half to one times the diameter of the bacterial cell body and had somewhat fuzzy, irregular borders. These capsules could be washed off easily by forcing a suspension of the bacteria through an 18-gauge needle. The isolate of N. meningitidis produced visible capsules when cultured with or without the SV. The isolates of N. sicca and N. subflava produced small, sharply defined capsules when grown in the presence of SV; however, these capsules were distinct from those of GC in that they were not removable by gentle mechanical means. So called virulent colony types (Ti) and the less virulent colony type (T4) (3) derived from GC strains 134 and 230, respectively, were tested by this system; both colony types produced large capsules. Each of the colony types was monitored for any change, after production of the capsule, by passage onto colony typing medium (3). No change in colony type was observed. Having established conditions under which GC produced capsules, we investigated the functional role such a capsule might play. The peripheral human blood leukocyte slide phagocytosis test of Swanson et al. (4) was adopted to determine whether capsules provided resist-
663
664
NOTES
INFECT. IMMUN.
TABLE 1. Capsule production by Neisseria species a Neisseria isolate
N. gonorrhoeae 230 120 108 134 u17 u19
No. of previous in vitro passes
>20 >20 10 5 3 3
N. meningiditis 138I
Capsule production
Without SV
With SV
0 0
++ ++ ++ ++
+
± +
++
+
++
++
++
N. subflava Three strains
5
0
±
N. sicca Three strains
5
0
+
Organisms were grown with or without "viridans streptococci" and observed in India ink-methylene blue preparations as described in the text, and the capsules were scored as follows: 0, no capsule; ±, equivocal capsule; +, maximum capsule thickness one-half the diameter of the blue-stained organism; + +, capsule thickness greater than one-half the diameter of the organism. a
FIG. 1. GC grown in the presence of SV were smeared onto a glass slide, and 1 drop of 5% methylene blue in ethanol and 1 drop of India ink were quickly added and mixed on the slide. A cover slip was put over the preparation, and the bacteria were observed at a magnification of xl ,OOO. The bacterial bodies stain blue, and the capsules appear as unstained halos against the black India ink.
to non-antibody-mediated phagocytosis. Unencapsulated GC were attached to or ingested by polymorphonuclear leukocytes (PMNs) quite easily in this system. In one experiment with unencapsulated GC 108 (T4), 88% of 400 PMNs counted had one or more organisms attached or ingested, with the averance
TABLE 2. Phagocytosis of encapsulated versus unencapsulated N. gonorrhoeae Gonococcal strain
Encapsulated a (%)
Unencapsulated a (%)
104 31 91 19 88 108 24 93 125 20 78 134 24 76 230 u12 26 92 a Percentage of PMNs with one or more gonococcal organisms either attached or ingested. Equal numbers of encapsulated or unencapsulated organisms (between 107 and 108 colony-forming units, depending on the experiment) suspended in 1 ml of medium 199 containing 0.01% bovine serum albumin (Armour Pharmaceutical Co., Chicago, Ill.) were added simultaneously to -5 x 106 human PMNs (isolated from a single volunteer) attached to Lab Tek tissue culture chamber slides (Miles Laboratories, Naperville, Ill.), and incubated with shaking at 370C for 20 min. Slides were then washed, fixed with methanol, and stained with Giemsa for 90 min.
age being 7.8 organisms per PMN. In contrast, only 19% of 400 cells counted could be found with GC attached or ingested in the leukocyte wells simultaneously incubated with encapsulated GC 108 (T4) (average, 1.6 organisms per PMN). Table 2 shows the results of six sets of paired experiments demonstrating the resistance of encapsulated GC to attachment and ingestion by leukocytes. That the elaboration of a capsule by GC is associated with increased resistance to phagocytosis suggests a functional role for the capsule. Efforts to chemically and immunologically characterize the capsule are underway, so that its true significance can be determined. We thank Kathyrn K. Yamada for technical assistance.
LITERATURE CITED 1. Casper, W. A. 1937. The preparation of the type-specific carbohydrates of gonococi. J. Immunol. 23:421-439. 2. Deacon, W. E., W. L. Peacock, E. M. Freeman, and A. Harris. 1959. Identification of Neisseria gonorrhoeae by means of fluorescent antibodies. Proc. Soc. Exp. Biol. Med. 101:322-325. 3. Kellogg, D. S., Jr., I. R. Cohen, L. C. Norins, A. L. Schroeter, and G. Reising. 1968. Neisseria gonorrhoeae. II. Colonial variation and pathogenicity during 35 months in vitro. J. Bacteriol. 96:596-605. 4. Swanson, J., E. Sparks, B. Zeligs, M. A. Siam, and C. Parrot. 1976. Studies on gonococcus infection. V. Observations on in vitro interactions of gonococci and human neutrophils. J. Infect. Dis. 133:441-447.
