Vol. 126, No. 2 Printed in U.S A.

JOURNAL OF BACTERIOLOGY, May 1976, p. 969-976 Copyright © 1976 American Society for Microbiology

Autolysis of Neisseria Gonorrhoeae THEODOR ELMROS,* LARS G. BURMAN, AND GUNNAR D. BLOOM Departments of Clinical Bacteriology* and Histology, University of Umed, S-901 87 Umed, Sweden Received for publication 31 July 1975

Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed.

Gonorrhea is today one of the most common bacterial infections in humans. A major problem in the control of gonococcal disease is the fragility of Neisseria gonorrhoeae, which makes transport of specimens and laboratory diagnostic work difficult. Many types of transport media have been evaluated. Still, the gonococci in 12 to 79% of positive specimens do not survive for 24 h at 22 C in the best of these media (13). It is also well known that unless daily subcultivation is performed in the laboratory a culture of gonococci can easily be lost. Dienes observed in 1940 that in aging colonies gonococci were transformed into "large bodies" (5), and he was later able to isolate L-forms from such colonies without the aid of penicillin (6). Furthermore, in continuous cultures 5 to 10% of the gonococci have been shown to be converted into similar "large bodies" (4). Many or possibly all bacteria that have a cell wall possess enzymes that break different bonds of the peptidoglycan component of the wall of the organism (9). That a peptidoglycan structurally related to that of Escherichia coli and other gram-negative bacteria also is present in the cell envelope of N. gonorrhoeae has recently been demonstrated (26). Peptidoglycan hydrolases appear to be most active at cell division (22). A physiological role in peptidoglycan expansion and, in particular, cell division has therefore been ascribed to these "autolytic" enzymes. One organism known for its extreme tendency to lyse spontaneously is Diplococcus pneumoniae (25). One reason for its lytic behavior could be the prolonged time necessary for cell separation that is typical for the pneumococcus, leading to its diplococcal cellular morphology. High autolytic enzyme activities might be present during a major part of the cell cycle, explaining the lytic behavior of the pneumococcus. The gonococcus similarly shows a

diplococcal morphology, suggesting a prolonged septation or cell separation phase. The purpose of the present work is to establish whether the fragility of N. gonorrhoeae is connected with lysis of the organism and whether lysis can be influenced by varying environmental conditions. Such information would increase our knowledge of the physiology of gonococci and could lead to improved diagnostic bacteriology in gonorrhea. MATERIALS AND METHODS Microorganisms. Colony types 1, 2, 3, and 4 of strain 82409/55 of N. gonorrhoeae were obtained from Alice Reyn, Copenhagen. The multiple antibiotic-resistant gonococcal strain Copenhagen V, N. pharyngitis (var. flavus, strain 4590) and N. meningitidis type B (strain 176/73) were from Statens Bakteriologiska Laboratorium, Stockholm, Sweden. The bacteria were stored at -60 C in GC medium base (Difco) plus supplement B and 20% glycerol and with agar omitted. Materials. The chemicals used were from Sigma Chemical Co., St. Louis, Mo., and from KEBO AB, Stockholm. Egg white lysozyme (grade I) was from Sigma. All glassware used was regularly washed with acid and carefully rinsed with glass-distilled water. Redistilled water was used for all solutions. Universal buffer (3) contains diethyl barbituric acid (0.028 M), citric acid (0.028 M), boric acid (0.028 M), and KH2PO4 (0.028 M). Cultivation. The bacteria were cultivated on GC medium base plus supplement B (GCMB) as solid medium at 36 C in a CO2 incubator (6% CO2). The colony types were examined after 20 to 22 h of incubation in a Zeiss plate microscope utilizing oblique transmitted light (16). In some experiments 1 part of medium 199 (Flow Laboratories) layered over 4 parts of GC agar base plus Isovitalex (GCAB, Baltimore Biological Laboratory) was used as liquid medium. In most experiments however, GCMB without agar (GCMB broth) was employed as liquid medium. Growth in liquid medium was performed at 36 C 969

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in a shaking bath, and optical density (OD) was recorded in a Klett-Summerson colorimeter with a W66 filter. An OD of 100 Klett units corresponds to 3 x 108 colony-forming units of type 4 gonococci per ml. Colony-forming units were determined after dilution in T2 buffer (17) using an automatic pipetting machine (Autochemist, Autochem, Lidingo, Sweden) and spreading on plates with the aid of a glass rod. The plates were incubated for 48 h before counting. To obtain log-phase cultures, about 108 cells were inoculated into 20 ml of medium in a 100-ml side-arm flask that was filled with 10% CO2 in air. The flask was sealed with a rubber stopper and incubated for about 5 h to reach a turbidity of 50 to 100 Klett units. Lysis experiments. Cells were harvested by centrifugation (2,000 x g) for 10 min at 4 C and were thereafter suspended at 0 C in the liquid to be tested. After zero-time absorbancy measurement, the suspension was placed in a 22 C water bath. Absorbancy was followed in a Beckman model 25 spectrophotometer at 540 nm. The initial absorbancy was about 0.15. Electron microscopy. All procedures were as described previously (20).

FIG. 1. Autolysis of N. gonorrhoeae (type 4). The bacteria were cultivated in medium 199 layered over GCAB, transferred to Tris-hydrochloride buffer (0.05 M, pH 7.2), and incubated at 22 C. Symbols: O, absorbancy at 540 nm; 0, absorbancy at 260 nm; *, absorbancy at 280 nm; *, colohy-forming units.

RESULTS Lysis of N. gonorrhoeae. When well-centrifuged log-phase type 4 gonococci were suspended in tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (0.05 M, pH 7.2), a continuous decrease in turbidity of the suspension and in colony-forming ability of the bacteria was observed (Fig. 1). Ultraviolet-absorbing material was simultaneously released into the suspending buffer. Thus, lysis of part of the bacterial population occurred, and most cells lost their colony-forming ability. Washing of the cells in buffers, saline, or distilled water before starting a lysis experiment resulted in a loss of about 20% of the turbidity of the cell suspension. Because a fraction of the cell population apparently lysed during washing, this procedure was not routinely applied. Hereafter, a spontaneous reduction of OD of a suspension of gonococci is referred to as autolysis. However, the turbidity measurements were grossly influenced by autoagglutination of the cells of certain colony types (2 > 1 > 3 > 4). Judging from radioactive isotope incorporation experiments and yields of cell envelope material, the massive clumping of type 2 cells reduced their OD by about two-thirds as compared to type 4 cells, which showed very little autoagglutination. Type 4 cells were therefore used in most experiments reported here. The shape of a lysis curve was influenced by small quantities of remaining growth medium that partly stabilized the cells. If the medium was carefully removed by decanting followed by suction and wiping the inside of the tube with a cotton tip, a more rapid lysis occurred compared

to decanting only. In all experiments reported here the former method was used. Gonococci harvested in the logarithmic growth phase or late-log phase showed a similar lytic behavior. On the other hand, when harvested 1 h after reaching stationary phase, the initial lysis was twice as rapid as log-phase cells. The antibiotic-sensitive strain 82409/55 was slightly more lytic than the multipleresistant strain Copenhagen V. There was no apparent difference in lytic tendency between cells belonging to colony types 1, 2, 3, and 4. These conclusions concerning differences in autolysis between colony types were based on the release of ult violet-absorbing material instead of OD which is influenced by autoagglutination as mentioned above. A somewhat higher lysis rate was observed for log-phase cells grown in GCMB broth than for cells grown in medium 199 layered over GCAB. A strain of N. meningitidis showed a slight initial lysis, and a strain of N. pharyngitis was not lytic in Tris-hydrochloride buffer. Electron microscopy. When studying autolysing N. gonorrhoeae by electron microscopy, a variation in lysis was seen among the cells (Fig. 2). A large fraction of the cells are ruptured. The cells at some stage of division appear distended, with a stretched outer membrane, and display a cytoplasm with varying degrees of decreased electron density. Such cells show abnormal bulging septal regions (see arrows in Fig. 2) instead of the sharp inward projecting septa seen in growing cells (Fig. 2, inset). Among the unlysed gonococci a certain number of small cells with normal outer membrane and

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Vol. 126, No. 2 Printed in U.S A. JOURNAL OF BACTERIOLOGY, May 1976, p. 969-976 Copyright © 1976 American Society for Microbiology Autolysis of Neis...
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