Biofilm Formation by Neisseria gonorrhoeae
http://www.100md.com
感染与免疫杂志 2005年第4期
Department of Microbiology, University of Iowa, Iowa City, Iowa
ABSTRACT
Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 μM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection.
INTRODUCTION
Neisseria gonorrhoeae is a human-adapted, gram-negative diplococcus that infects the human male and female reproductive tracts. N. gonorrhoeae infections in women frequently go unnoticed. This can eventually lead to serious upper genital tract infections which ultimately can lead to infertility (13). Currently, no studies have discussed the ability of N. gonorrhoeae to produce biofilms. Bacterial biofilms have been defined as communities of bacteria intimately associated with each other and included within an exopolymer matrix. These biological units exhibit their own properties, which are quite different from those shown by the single species in planktonic form (15). Numerous bacterial species are capable of producing biofilms. Biofilms confer a number of survival advantages to the bacteria, including increased resistance to antimicrobial agents (7, 18).
Our interest in the capability of N. gonorrhoeae to form a biofilm came about by observations made in our laboratory during 4- and 8-day infections of primary human urethral and cervical epithelial cells (8, 12). Those studies showed that the gonococcus was forming microcolonies on these surfaces, and eventually these transitioned into structures that resembled bacterial biofilms.
The purpose of this study was twofold. The first objective was to verify that N. gonorrhoeae can produce a biofilm both in biofilm chambers and over primary human genital tract epithelial cells in culture. The second objective was to gain information about the structure of the gonococcal biofilm. To accomplish this, we established gonococcal infections in continuous-flow chambers and on primary human genital tract epithelial cells. Light and electron microscopic analyses indicated that the gonococcus could form a biofilm on these surfaces and that the ultrastructure resembled that previously seen with other bacteria, with the exception that the biofilm was interlaced with what appeared to be membranous structures surrounding the organisms. Lectin and antibody analyses indicated that the biofilm sugars were similar to those that were expressed as terminal saccharides of gonococcal lipooligosaccharide (LOS).
MATERIALS AND METHODS
Bacteria and culture conditions. N. gonorrhoeae strains 1291, FA1090, and MS11, used in this study, are clinical isolates. These strains were reconstituted from frozen stock cultures and propagated at 37°C with 5% CO2 on GC agar (Difco, Detroit, Mich.) supplemented with 10 ml of IsoVitaleX (Becton-Dickinson, Franklin Lakes, N.J.) per liter. These N. gonorrhoeae strains also were transformed with pGFP (pLES98 containing gfp) to express green fluorescent protein (GFP). The plasmid pLES98 was a gift from V. Clark. The strain number followed by pGFP in brackets is used to designate these transformed strains.
Biofilm growth in a continuous-flow chamber. Gonococcal strains were grown in a continuous-flow chamber identical to that described by Davies et al. (5). RPMI 1640 medium (Gibco, Grand Island, N.Y.) containing 100 μM sodium pyruvate (Gibco), 20 ml of hypoxanthine (370 μM) and uracil (450 μM) per liter, 100 μM sodium nitrite, and 1% IsoVitaleX was prepared. In some experiments, 20 μM CMP-N-acetylneuraminic acid (CMP-Neu5Ac) was added to the medium. This solution was diluted 1:10 with sterile phosphate-buffered saline (PBS) and was used in the continuous-flow experiments. To inoculate the chamber, 1 ml of N. gonorrhoeae culture, grown to a density of 108 organisms/ml, was placed in the chamber and left for 1 h. The flow was then started at 150 μl/min. The biofilm was formed in a 37°C environmental incubator and continuously perfused over the duration of the experiment. At the end of that time period, the effluent was cultured to assure that the culture purity was maintained. Digital biofilm images were then collected.
Laser scanning confocal microscopy of continuous-flow chambers and cultured epithelial cells. Confocal images of biofilms in continuous-flow chambers and on primary human cervical epithelial cells in culture were obtained with a Bio-Rad MRC-1024 scanning confocal microscope as previously described (8). The chambers were viewed in situ under the confocal microscope. Cervical epithelial cells were grown on collagen-coated glass coverslips in a 24-well plate. Infections were performed in the chambers, and at various time points the coverslips were removed from the chambers and viewed with the confocal microscope after appropriate staining.
Viability staining of bacteria from continuous-flow chamber biofilms. To evaluate the viability of bacteria present within the biofilm matrix, N. gonorrhoeae 1291 was grown in a flow chamber as described above. After 4 days, the flow chamber was carefully disconnected. The Live/Dead BacLight bacterial viability kit (Molecular Probes, Eugene, Oreg.) was used to visualize live and dead bacteria within the biofilm. Briefly, SYTO 9 (component A) and propidium iodide (component B) were mixed at a 1:1 ratio. Three microliters of the viability stain was added to 1 ml of PBS. Medium in the chamber was aseptically replaced with the stain-PBS mixture. The chamber was incubated for 15 min at 37°C. One milliliter of sterile PBS was then added to the chamber to flush away excess stain. Biofilm bacteria within the chamber were immediately visualized with a Zeiss 510 laser scanning confocal microscope at a magnification of x10. The resulting images were compiled as cross-sections of a z series.
Fixation of biofilm samples for microscopy. All samples used for microscopy were grown in biofilm chambers in RPMI 1640 medium as described above. In all experiments other than the live/dead studies, a 1-ml mixture of 4% paraformaldehyde and 5% dimethyl sulfoxide was infused into the chamber at the end of the growth period and allowed to fix overnight. Samples were then embedded in situ in OCT resin (Sakura Finetek USA, Inc., Torrance, Calif.) on the coverslip surface upon which they were formed. After hardening, the coverslip was removed by freezing the sample in liquid nitrogen and shattering the glass, leaving the biofilm within the OCT resin. The biofilm was then cut into 1-μm-thick sections. N. gonorrhoeae strain 1291 was used for microscopic analyses unless otherwise noted.
Microscopy. OCT cryosections were incubated with hematoxylin and eosin stains to visualize the biofilm matrix. Images were viewed with an Olympus light microscope.
Biofilm samples from continuous-flow chambers were prepared for transmission electron microscopy (TEM) by using perfluorocarbon methods to minimize the extraction of water (22). Samples were embedded in Epon resin, sectioned, and viewed on an H-7000 instrument (Hitachi, Mountain View, Calif.) at a 75-kV accelerating voltage as previously described (12).
Biofilms grown on primary human cervical epithelial cells were processed for scanning electron microscopy (SEM) and viewed with a Hitachi S-4000 scanning electron microscope (8). Briefly, coverslips were fixed in a 2% osmium tetroxide-perfluorocarbon solution for 2 h, dehydrated with three 100% ethanol washes, and dried with hexamethyldisilazane (22) to preserve biofilm formation. Processed coverslips were then mounted onto stubs with colloidal silver and were sputter coated with gold palladium.
Biofilm samples grown in the presence of CMP-Neu5Ac were cultured (with gentle agitation) on glass coverslips, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), and rinsed in double-distilled H2O immediately before plunge-freezing in liquid nitrogen. Frozen specimens were introduced into an Emitech K1250 cryogenic system preparation chamber for radiant heat surface etching (a tungsten filament was heated by 20 A of current for 2 min). The uncoated samples were then introduced into and imaged with a Hitachi S-4000 cold cathode field emission scanning electron microscope (C-FESEM) (9, 21) All of the microscopes used in these studies are located at the University of Iowa Central Microscopy Research Facility (Iowa City).
Lectin analysis of biofilms. The OCT resin sections were studied by fluorescence microscopy with the fluorescein-conjugated lectins Maackia amurensis, Sambucus nigra, succinylated wheat germ, soybean agglutinin, and Amaryllis (all from EY Laboratories, San Mateo, Calif.) as previously described (10).
Antibody analysis of biofilms. OCT embedded sections were incubated with monoclonal antibodies (MAbs) 6B4 (murine immunoglobulin M [IgM]) and 2C3 (murine IgG). Anti-murine IgM-fluorescein isothiocyanate and anti-IgG-tetramethyl rhodamine isocyanate were used as secondary antibodies. Images of the samples were taken with the Bio-Rad MRC-1024 laser scanning confocal viewing system.
Gonococcal biofilm formation on primary human cervical epithelial cells. Surgical biopsies derived from the ecto- and the endocervix that were used to seed primary cervical epithelial cell systems were procured and maintained as described previously (5) in defined keratinocyte serum-free medium (Life Technologies, Rockville, Md.). Primary cervical epithelial cells were grown on coverslips as previously described by Edwards et al. (8). Once the cells were confluent, N. gonorrhoeae was added to the cell monolayer at a multiplicity of infection of 100. Infected cell layers were then incubated in a 37°C incubator with 5% CO2 for 4 or 8 days. Samples were then prepared for SEM. In another set of experiments, samples were infected with N. gonorrhoeae 1291[pGFP], fixed, and then stained with ethidium bromide. These samples were viewed with the Bio-Rad MRC-1024 laser scanning confocal viewing system.
RESULTS
Continuous-flow chamber studies. To test the hypothesis that gonococci could form biofilms, we performed studies in which continuous-flow chambers (7) were infected for 1 h with 108 piliated strain 1291 gonococci. At the end of that time period, the chamber was continually perfused at a flow rate of 150 μl/min with supplemented RPMI 1640 containing 10 μM sodium nitrite. Nitrite was initially added to ensure gonococcal survival in the relatively low-oxygen environment of the flow cell. Figure 1A shows the result of a typical 5-day flow chamber study with N. gonorrhoeae strain 1291. As can be seen, approximately 80% of the coverslip surface is covered with what appears to be a biofilm. Flow eddies can be seen where the biofilm had been washed off the coverslip by the flowing medium. Attempts to reduce the flow rate in the chamber resulted in increased biofilm formation and obstruction of the chamber flow. We performed live/dead staining immediately after the termination of the experiment (Fig. 1B). The clusters of organisms nearest the surface of the glass coverslip appeared to be nonviable, while those closer to the medium flow stream appeared to be viable. Gonococcal strains MS11 and FA1090 were also studied in the flow chamber and were found to be capable of forming biofilms (data not shown). We assumed that within the chamber the gonococcus would need nitrite as an electron acceptor, as we believed that the chamber would constitute a microaerophilic environment. Our studies indicated that the gonococcus would grow in the flow chamber in the absence of nitrite. Figures 1C and E and 1D and F show confocal analyses of a biofilm produced by N. gonorrhoeae strain 1291 in the continuous-flow chamber over 4 days in the presence (final concentration, 10 μM) and absence of nitrite, respectively. These studies suggested that the biofilm topography might vary under these different conditions. Biofilm formation in the absence of nitrite suggested that the chamber environment was not microaerophilic or, alternatively, that other factors such as cytochrome oxidase might facilitate cell growth under microaerophilic conditions.
Microscopic analyses. We performed initial light microscopy studies of stained cryosections of biofilms taken from coverslips. To maintain the in situ conformation of the biofilm, the samples were cryopreserved in dimethyl sulfoxide, embedded in OCT, snap frozen, and sectioned on a cryomicrotome. As can be seen in Fig. 2A, in a hematoxylin- and eosin-stained section, the biofilm had numerous water channels. In addition, the organisms within the biofilm were surrounded by what appears to be a pink-staining matrix (Fig. 2A).
We performed C-FESEM on a 3-day N. gonorrhoeae 1291 biofilm. Figure 2B and C and shows organisms embedded in a continuous matrix. Demarcated water channels could be clearly seen within the biofilm. In addition, what appeared to be membrane-like structures spanned areas of the biofilm (Fig. 2C). Transmission electron microscopy of biofilm samples fixed in perfluorocarbon also revealed extensive membrane-like structures enclosing organisms within the biofilm, with traces of what appeared to be residual matrix material (Fig. 3A and B). TEM analysis of the apical portion of the biofilm showed a mass of membrane-like structures covering organisms (Fig. 3C). Figure 3D shows an immunoelectron micrograph of a gonococcal biofilm on a primary human urethral epithelial cell. Similar membrane-like structures were seen within these biofilms. These membranes strained with monoclonal antibody 6B4, which recognizes a lactosamine epitope known to be present on gonococcal LOS (1).
Lectin analysis of the biofilm. We also performed lectin-binding studies with frozen, OCT-embedded biofilm formed in the absence of CMP-Neu5Ac (Fig. 4). Sections were labeled with the following fluorescein-conjugated lectins: succinylated wheat germ agglutinin (primary specificity, N-acetylglucosamine), S. nigra (primary specificity, Neu5Ac 2-6Gal), M. amurensis (primary specificity, Neu5Ac 3-6Gal), Amaryllis (primary specificity, mannose), and soybean agglutinin (primary specificity, -linked N-acetylgalactosamine; secondary specificity, galactose). The only lectin that bound to the biofilm was soybean agglutinin. This suggested that the terminal sugar on the biofilm matrix when grown in the absence of CMP-Neu5Ac was either a -linked N-acetylgalactosamine or a galactose. To test whether Neu5Ac is incorporated into the biofilm, CMP-Neu5Ac (10 μM) was added to the growth medium. Lectin analysis demonstrated that soybean agglutinin failed to bind to the biofilm formed in the presence of CMP-Neu5Ac and that binding was restored after neuraminidase treatment of the biofilm (Fig. 5). M. amurensis lectin bound to the sialylated biofilm, and this binding was eliminated by treatment with neuraminidase. These studies indicated that NeuAc could be incorporated into the biofilm if the substrate CMP-Neu5Ac was available.
Antibody analysis of the biofilm. To confirm the lectin studies, biofilm embedded in OCT was incubated with MAbs 6B4 (murine IgM) and 2C3 (murine IgG). Monoclonal antibody 6B4 recognizes a Gal1-4GlcNAc epitope (which is present also on strain 1291 LOS), and MAb 2C3 binds to the H.8 protein of pathogenic Neisseria spp. Anti-murine IgM-fluorescein isothiocyanate and anti-IgG-tetramethyl rhodamine isocyanate were used as secondary antibodies. If the biofilm had a terminal lactosamine structure (Gal1-4GlcNAc), we predicted that we might detect green fluorescence surrounding yellow-orange-staining organisms. Figure 6 shows the results of this experiment, which demonstrates this and indicates that the biofilm matrix itself binds MAb 6B4, as areas in which 6B4 binding occurs without colocalization with 2C3-labeled bacteria can be seen. Similar studies were performed on biofilm formed in the presence of CMP-Neu5Ac. These studies demonstrated that MAb 6B4 failed to bind the sialylated biofilm but that after treatment with neuraminidase, MAb 6B4 did bind to the biofilm (data not shown). Thus, the conclusions from the lectin and monoclonal antibody studies indicated that predominate terminal sugars of the biofilm in the absence and presence of CMP-Neu5Ac are lactosamine and sialyllactosamine, respectively.
Gonococcal biofilm formation on primary human cervical epithelial cells. Our hypothesis that gonococci produced biofilms originated when we observed microcolonies and what appeared to be biofilms on cultured primary human genital tract cells after 3- and 4-day infections. Figure 7 shows a confocal study of human primary cervical epithelial cells infected with N. gonorrhoeae strain 1291[pGFP]. A biofilm layer extending approximately 20 to 30 μm above the primary human cervical epithelial cells in what appeared to be tower-like formations was seen. The epithelial cell layer remained intact during these experiments, and trypan blue exclusion studies demonstrated that over 95% of the infected epithelial cells were viable at the termination of the experiment at 4 and 8 days. SEM analysis of 4- and 8-day gonococcal infections revealed biofilm formation over cervical epithelial cells that covered almost the entire epithelial cell surface by day 8 after initiation of infection (Fig. 8).
DISCUSSION
These studies demonstrated that N. gonorrhoeae could form a biofilm in a continuous-flow chamber and over primary human genital tract epithelial cells in culture. Biofilms are complex communities of bacteria that develop in diverse environments (11). They are dynamic structures in which cells switch from a weak interaction with the surface to an almost permanent binding through extracellular polymers. In addition, the bacteria form channels and pores to provide nutrients to bacteria within the biofilm structure. The maintenance of a biofilm is attributed to the development and preservation of the exopolysaccharide matrix (4). More than 300 proteins that are not detected in planktonic bacteria can be detected in bacteria from mature biofilms (20). These proteins fall into the functional classes of metabolism, phospholipid and lipopolysaccharide biosynthesis, membrane transport and secretion, and adaptation and protective mechanisms. In addition, biofilm bacteria are considered to be in the stationary phase of growth, partly because of the accumulation of acylhomoserine lactone within cell clusters (23). Some bacteria eventually detach from the mature biofilm to enter the surrounding fluid phase. Detachment is a physiologically regulated event in which bacteria will release from the biofilm as a planktonic organism to move on to attach to other surfaces, where they initiate biofilm development (16). Many different mechanisms may contribute to the detachment process. O'Toole and Kolter (17) demonstrated that starvation may lead to detachment by an unknown mechanism. Streptococcus mutans produces a surface protein-releasing enzyme that mediates its release from biofilms (14). A possible trigger for the release of this matrix-degrading enzyme could be cell density. In addition, the presence of homoserine lactones may cause biofilm reduction, as has been demonstrated with Rhodobacter sphaeroides (17, 19).
These studies have utilized several microscopic approaches to elucidate the structure of the biofilm produced by N. gonorrhoeae. Cryosections viewed by light microscopy indicated that organisms were surrounded by a matrix (Fig. 2A). Cryofield emission scanning electron microscopy and TEM have shown that this biofilm consists of membranous structures that appeared to be surrounding gonococci that may also be encased in a matrix. Binding studies with lectins and monoclonal antibody 6B4 showed that LOS-like structures predominate on the biofilm and that these structures could be identified separated from organisms within the biofilm (Fig. 3B and 6). These membranous extensions are interesting. They can reach 10 to 15 μm in length (Fig. 3A and B). We presume that they are derived from the bacterial outer membrane, which is shed from the gonococcus in the form of blebs. Our studies have shown that human primary genital tract epithelial cells can serve as surfaces for gonococcal biofilm formation with minimal, if any, effect on cell viability. This may reflect the ability of the gonococcus to induce antiapoptotic factors in these cells (2, 3).
Immunoelectron microscopy experiments with primary urethral epithelial cells show membrane structures resembling those seen in Fig. 3A. These structures stained heavily with monoclonal antibody 6B4. It is interesting to speculate that these membranous structures are derived in large part from the fusions of blebs shed by the gonococcus and the meningococcus during growth (6). These may encase the gonococci within sac-like structures into which a matrix is released. A recent study of biofilms produced by 39 Neisseria meningitidis strains showed that 30% of the carriage isolates and 12.5% of the invasive disease isolates formed biofilms in microtiter wells (25). Generally, the more hydrophobic the surface of the organism, the more likely it was to form a biofilm, and encapsulation inhibited biofilm formation. These studies support the concept that hydrophobic interactions of surfaces with pathogenic Neisseria spp. enhance their ability to form biofilms. The present study lends support to the theory that the membranous bacterially derived structures may facilitate biofilm formation.
A number of previous studies have demonstrated that gonococci can persist in an asymptomatic state in the female genital tract (13). In addition, antibiotic resistance to a broad range of agents has become a major concern with the management of gonococcal infections (24). The ability of this organism to form a biofilm on human cells, particularly in the female genital tract, may be a factor in both of these consequences of gonococcal infection. Future studies will be directed at studying the nature of the biofilm matrix and the role of biofilms during infection of cervical epithelial cells.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant AI AI45728 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (to M.A.A).
We acknowledge the staff of the Central Microscopy Research Facility at the University of Iowa.
L.L.G. and J.L.E. contributed equally to the work described in this paper.
Present address: Columbus Children's Research Institute, Columbus, OH 43205.
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ABSTRACT
Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 μM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection.
INTRODUCTION
Neisseria gonorrhoeae is a human-adapted, gram-negative diplococcus that infects the human male and female reproductive tracts. N. gonorrhoeae infections in women frequently go unnoticed. This can eventually lead to serious upper genital tract infections which ultimately can lead to infertility (13). Currently, no studies have discussed the ability of N. gonorrhoeae to produce biofilms. Bacterial biofilms have been defined as communities of bacteria intimately associated with each other and included within an exopolymer matrix. These biological units exhibit their own properties, which are quite different from those shown by the single species in planktonic form (15). Numerous bacterial species are capable of producing biofilms. Biofilms confer a number of survival advantages to the bacteria, including increased resistance to antimicrobial agents (7, 18).
Our interest in the capability of N. gonorrhoeae to form a biofilm came about by observations made in our laboratory during 4- and 8-day infections of primary human urethral and cervical epithelial cells (8, 12). Those studies showed that the gonococcus was forming microcolonies on these surfaces, and eventually these transitioned into structures that resembled bacterial biofilms.
The purpose of this study was twofold. The first objective was to verify that N. gonorrhoeae can produce a biofilm both in biofilm chambers and over primary human genital tract epithelial cells in culture. The second objective was to gain information about the structure of the gonococcal biofilm. To accomplish this, we established gonococcal infections in continuous-flow chambers and on primary human genital tract epithelial cells. Light and electron microscopic analyses indicated that the gonococcus could form a biofilm on these surfaces and that the ultrastructure resembled that previously seen with other bacteria, with the exception that the biofilm was interlaced with what appeared to be membranous structures surrounding the organisms. Lectin and antibody analyses indicated that the biofilm sugars were similar to those that were expressed as terminal saccharides of gonococcal lipooligosaccharide (LOS).
MATERIALS AND METHODS
Bacteria and culture conditions. N. gonorrhoeae strains 1291, FA1090, and MS11, used in this study, are clinical isolates. These strains were reconstituted from frozen stock cultures and propagated at 37°C with 5% CO2 on GC agar (Difco, Detroit, Mich.) supplemented with 10 ml of IsoVitaleX (Becton-Dickinson, Franklin Lakes, N.J.) per liter. These N. gonorrhoeae strains also were transformed with pGFP (pLES98 containing gfp) to express green fluorescent protein (GFP). The plasmid pLES98 was a gift from V. Clark. The strain number followed by pGFP in brackets is used to designate these transformed strains.
Biofilm growth in a continuous-flow chamber. Gonococcal strains were grown in a continuous-flow chamber identical to that described by Davies et al. (5). RPMI 1640 medium (Gibco, Grand Island, N.Y.) containing 100 μM sodium pyruvate (Gibco), 20 ml of hypoxanthine (370 μM) and uracil (450 μM) per liter, 100 μM sodium nitrite, and 1% IsoVitaleX was prepared. In some experiments, 20 μM CMP-N-acetylneuraminic acid (CMP-Neu5Ac) was added to the medium. This solution was diluted 1:10 with sterile phosphate-buffered saline (PBS) and was used in the continuous-flow experiments. To inoculate the chamber, 1 ml of N. gonorrhoeae culture, grown to a density of 108 organisms/ml, was placed in the chamber and left for 1 h. The flow was then started at 150 μl/min. The biofilm was formed in a 37°C environmental incubator and continuously perfused over the duration of the experiment. At the end of that time period, the effluent was cultured to assure that the culture purity was maintained. Digital biofilm images were then collected.
Laser scanning confocal microscopy of continuous-flow chambers and cultured epithelial cells. Confocal images of biofilms in continuous-flow chambers and on primary human cervical epithelial cells in culture were obtained with a Bio-Rad MRC-1024 scanning confocal microscope as previously described (8). The chambers were viewed in situ under the confocal microscope. Cervical epithelial cells were grown on collagen-coated glass coverslips in a 24-well plate. Infections were performed in the chambers, and at various time points the coverslips were removed from the chambers and viewed with the confocal microscope after appropriate staining.
Viability staining of bacteria from continuous-flow chamber biofilms. To evaluate the viability of bacteria present within the biofilm matrix, N. gonorrhoeae 1291 was grown in a flow chamber as described above. After 4 days, the flow chamber was carefully disconnected. The Live/Dead BacLight bacterial viability kit (Molecular Probes, Eugene, Oreg.) was used to visualize live and dead bacteria within the biofilm. Briefly, SYTO 9 (component A) and propidium iodide (component B) were mixed at a 1:1 ratio. Three microliters of the viability stain was added to 1 ml of PBS. Medium in the chamber was aseptically replaced with the stain-PBS mixture. The chamber was incubated for 15 min at 37°C. One milliliter of sterile PBS was then added to the chamber to flush away excess stain. Biofilm bacteria within the chamber were immediately visualized with a Zeiss 510 laser scanning confocal microscope at a magnification of x10. The resulting images were compiled as cross-sections of a z series.
Fixation of biofilm samples for microscopy. All samples used for microscopy were grown in biofilm chambers in RPMI 1640 medium as described above. In all experiments other than the live/dead studies, a 1-ml mixture of 4% paraformaldehyde and 5% dimethyl sulfoxide was infused into the chamber at the end of the growth period and allowed to fix overnight. Samples were then embedded in situ in OCT resin (Sakura Finetek USA, Inc., Torrance, Calif.) on the coverslip surface upon which they were formed. After hardening, the coverslip was removed by freezing the sample in liquid nitrogen and shattering the glass, leaving the biofilm within the OCT resin. The biofilm was then cut into 1-μm-thick sections. N. gonorrhoeae strain 1291 was used for microscopic analyses unless otherwise noted.
Microscopy. OCT cryosections were incubated with hematoxylin and eosin stains to visualize the biofilm matrix. Images were viewed with an Olympus light microscope.
Biofilm samples from continuous-flow chambers were prepared for transmission electron microscopy (TEM) by using perfluorocarbon methods to minimize the extraction of water (22). Samples were embedded in Epon resin, sectioned, and viewed on an H-7000 instrument (Hitachi, Mountain View, Calif.) at a 75-kV accelerating voltage as previously described (12).
Biofilms grown on primary human cervical epithelial cells were processed for scanning electron microscopy (SEM) and viewed with a Hitachi S-4000 scanning electron microscope (8). Briefly, coverslips were fixed in a 2% osmium tetroxide-perfluorocarbon solution for 2 h, dehydrated with three 100% ethanol washes, and dried with hexamethyldisilazane (22) to preserve biofilm formation. Processed coverslips were then mounted onto stubs with colloidal silver and were sputter coated with gold palladium.
Biofilm samples grown in the presence of CMP-Neu5Ac were cultured (with gentle agitation) on glass coverslips, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), and rinsed in double-distilled H2O immediately before plunge-freezing in liquid nitrogen. Frozen specimens were introduced into an Emitech K1250 cryogenic system preparation chamber for radiant heat surface etching (a tungsten filament was heated by 20 A of current for 2 min). The uncoated samples were then introduced into and imaged with a Hitachi S-4000 cold cathode field emission scanning electron microscope (C-FESEM) (9, 21) All of the microscopes used in these studies are located at the University of Iowa Central Microscopy Research Facility (Iowa City).
Lectin analysis of biofilms. The OCT resin sections were studied by fluorescence microscopy with the fluorescein-conjugated lectins Maackia amurensis, Sambucus nigra, succinylated wheat germ, soybean agglutinin, and Amaryllis (all from EY Laboratories, San Mateo, Calif.) as previously described (10).
Antibody analysis of biofilms. OCT embedded sections were incubated with monoclonal antibodies (MAbs) 6B4 (murine immunoglobulin M [IgM]) and 2C3 (murine IgG). Anti-murine IgM-fluorescein isothiocyanate and anti-IgG-tetramethyl rhodamine isocyanate were used as secondary antibodies. Images of the samples were taken with the Bio-Rad MRC-1024 laser scanning confocal viewing system.
Gonococcal biofilm formation on primary human cervical epithelial cells. Surgical biopsies derived from the ecto- and the endocervix that were used to seed primary cervical epithelial cell systems were procured and maintained as described previously (5) in defined keratinocyte serum-free medium (Life Technologies, Rockville, Md.). Primary cervical epithelial cells were grown on coverslips as previously described by Edwards et al. (8). Once the cells were confluent, N. gonorrhoeae was added to the cell monolayer at a multiplicity of infection of 100. Infected cell layers were then incubated in a 37°C incubator with 5% CO2 for 4 or 8 days. Samples were then prepared for SEM. In another set of experiments, samples were infected with N. gonorrhoeae 1291[pGFP], fixed, and then stained with ethidium bromide. These samples were viewed with the Bio-Rad MRC-1024 laser scanning confocal viewing system.
RESULTS
Continuous-flow chamber studies. To test the hypothesis that gonococci could form biofilms, we performed studies in which continuous-flow chambers (7) were infected for 1 h with 108 piliated strain 1291 gonococci. At the end of that time period, the chamber was continually perfused at a flow rate of 150 μl/min with supplemented RPMI 1640 containing 10 μM sodium nitrite. Nitrite was initially added to ensure gonococcal survival in the relatively low-oxygen environment of the flow cell. Figure 1A shows the result of a typical 5-day flow chamber study with N. gonorrhoeae strain 1291. As can be seen, approximately 80% of the coverslip surface is covered with what appears to be a biofilm. Flow eddies can be seen where the biofilm had been washed off the coverslip by the flowing medium. Attempts to reduce the flow rate in the chamber resulted in increased biofilm formation and obstruction of the chamber flow. We performed live/dead staining immediately after the termination of the experiment (Fig. 1B). The clusters of organisms nearest the surface of the glass coverslip appeared to be nonviable, while those closer to the medium flow stream appeared to be viable. Gonococcal strains MS11 and FA1090 were also studied in the flow chamber and were found to be capable of forming biofilms (data not shown). We assumed that within the chamber the gonococcus would need nitrite as an electron acceptor, as we believed that the chamber would constitute a microaerophilic environment. Our studies indicated that the gonococcus would grow in the flow chamber in the absence of nitrite. Figures 1C and E and 1D and F show confocal analyses of a biofilm produced by N. gonorrhoeae strain 1291 in the continuous-flow chamber over 4 days in the presence (final concentration, 10 μM) and absence of nitrite, respectively. These studies suggested that the biofilm topography might vary under these different conditions. Biofilm formation in the absence of nitrite suggested that the chamber environment was not microaerophilic or, alternatively, that other factors such as cytochrome oxidase might facilitate cell growth under microaerophilic conditions.
Microscopic analyses. We performed initial light microscopy studies of stained cryosections of biofilms taken from coverslips. To maintain the in situ conformation of the biofilm, the samples were cryopreserved in dimethyl sulfoxide, embedded in OCT, snap frozen, and sectioned on a cryomicrotome. As can be seen in Fig. 2A, in a hematoxylin- and eosin-stained section, the biofilm had numerous water channels. In addition, the organisms within the biofilm were surrounded by what appears to be a pink-staining matrix (Fig. 2A).
We performed C-FESEM on a 3-day N. gonorrhoeae 1291 biofilm. Figure 2B and C and shows organisms embedded in a continuous matrix. Demarcated water channels could be clearly seen within the biofilm. In addition, what appeared to be membrane-like structures spanned areas of the biofilm (Fig. 2C). Transmission electron microscopy of biofilm samples fixed in perfluorocarbon also revealed extensive membrane-like structures enclosing organisms within the biofilm, with traces of what appeared to be residual matrix material (Fig. 3A and B). TEM analysis of the apical portion of the biofilm showed a mass of membrane-like structures covering organisms (Fig. 3C). Figure 3D shows an immunoelectron micrograph of a gonococcal biofilm on a primary human urethral epithelial cell. Similar membrane-like structures were seen within these biofilms. These membranes strained with monoclonal antibody 6B4, which recognizes a lactosamine epitope known to be present on gonococcal LOS (1).
Lectin analysis of the biofilm. We also performed lectin-binding studies with frozen, OCT-embedded biofilm formed in the absence of CMP-Neu5Ac (Fig. 4). Sections were labeled with the following fluorescein-conjugated lectins: succinylated wheat germ agglutinin (primary specificity, N-acetylglucosamine), S. nigra (primary specificity, Neu5Ac 2-6Gal), M. amurensis (primary specificity, Neu5Ac 3-6Gal), Amaryllis (primary specificity, mannose), and soybean agglutinin (primary specificity, -linked N-acetylgalactosamine; secondary specificity, galactose). The only lectin that bound to the biofilm was soybean agglutinin. This suggested that the terminal sugar on the biofilm matrix when grown in the absence of CMP-Neu5Ac was either a -linked N-acetylgalactosamine or a galactose. To test whether Neu5Ac is incorporated into the biofilm, CMP-Neu5Ac (10 μM) was added to the growth medium. Lectin analysis demonstrated that soybean agglutinin failed to bind to the biofilm formed in the presence of CMP-Neu5Ac and that binding was restored after neuraminidase treatment of the biofilm (Fig. 5). M. amurensis lectin bound to the sialylated biofilm, and this binding was eliminated by treatment with neuraminidase. These studies indicated that NeuAc could be incorporated into the biofilm if the substrate CMP-Neu5Ac was available.
Antibody analysis of the biofilm. To confirm the lectin studies, biofilm embedded in OCT was incubated with MAbs 6B4 (murine IgM) and 2C3 (murine IgG). Monoclonal antibody 6B4 recognizes a Gal1-4GlcNAc epitope (which is present also on strain 1291 LOS), and MAb 2C3 binds to the H.8 protein of pathogenic Neisseria spp. Anti-murine IgM-fluorescein isothiocyanate and anti-IgG-tetramethyl rhodamine isocyanate were used as secondary antibodies. If the biofilm had a terminal lactosamine structure (Gal1-4GlcNAc), we predicted that we might detect green fluorescence surrounding yellow-orange-staining organisms. Figure 6 shows the results of this experiment, which demonstrates this and indicates that the biofilm matrix itself binds MAb 6B4, as areas in which 6B4 binding occurs without colocalization with 2C3-labeled bacteria can be seen. Similar studies were performed on biofilm formed in the presence of CMP-Neu5Ac. These studies demonstrated that MAb 6B4 failed to bind the sialylated biofilm but that after treatment with neuraminidase, MAb 6B4 did bind to the biofilm (data not shown). Thus, the conclusions from the lectin and monoclonal antibody studies indicated that predominate terminal sugars of the biofilm in the absence and presence of CMP-Neu5Ac are lactosamine and sialyllactosamine, respectively.
Gonococcal biofilm formation on primary human cervical epithelial cells. Our hypothesis that gonococci produced biofilms originated when we observed microcolonies and what appeared to be biofilms on cultured primary human genital tract cells after 3- and 4-day infections. Figure 7 shows a confocal study of human primary cervical epithelial cells infected with N. gonorrhoeae strain 1291[pGFP]. A biofilm layer extending approximately 20 to 30 μm above the primary human cervical epithelial cells in what appeared to be tower-like formations was seen. The epithelial cell layer remained intact during these experiments, and trypan blue exclusion studies demonstrated that over 95% of the infected epithelial cells were viable at the termination of the experiment at 4 and 8 days. SEM analysis of 4- and 8-day gonococcal infections revealed biofilm formation over cervical epithelial cells that covered almost the entire epithelial cell surface by day 8 after initiation of infection (Fig. 8).
DISCUSSION
These studies demonstrated that N. gonorrhoeae could form a biofilm in a continuous-flow chamber and over primary human genital tract epithelial cells in culture. Biofilms are complex communities of bacteria that develop in diverse environments (11). They are dynamic structures in which cells switch from a weak interaction with the surface to an almost permanent binding through extracellular polymers. In addition, the bacteria form channels and pores to provide nutrients to bacteria within the biofilm structure. The maintenance of a biofilm is attributed to the development and preservation of the exopolysaccharide matrix (4). More than 300 proteins that are not detected in planktonic bacteria can be detected in bacteria from mature biofilms (20). These proteins fall into the functional classes of metabolism, phospholipid and lipopolysaccharide biosynthesis, membrane transport and secretion, and adaptation and protective mechanisms. In addition, biofilm bacteria are considered to be in the stationary phase of growth, partly because of the accumulation of acylhomoserine lactone within cell clusters (23). Some bacteria eventually detach from the mature biofilm to enter the surrounding fluid phase. Detachment is a physiologically regulated event in which bacteria will release from the biofilm as a planktonic organism to move on to attach to other surfaces, where they initiate biofilm development (16). Many different mechanisms may contribute to the detachment process. O'Toole and Kolter (17) demonstrated that starvation may lead to detachment by an unknown mechanism. Streptococcus mutans produces a surface protein-releasing enzyme that mediates its release from biofilms (14). A possible trigger for the release of this matrix-degrading enzyme could be cell density. In addition, the presence of homoserine lactones may cause biofilm reduction, as has been demonstrated with Rhodobacter sphaeroides (17, 19).
These studies have utilized several microscopic approaches to elucidate the structure of the biofilm produced by N. gonorrhoeae. Cryosections viewed by light microscopy indicated that organisms were surrounded by a matrix (Fig. 2A). Cryofield emission scanning electron microscopy and TEM have shown that this biofilm consists of membranous structures that appeared to be surrounding gonococci that may also be encased in a matrix. Binding studies with lectins and monoclonal antibody 6B4 showed that LOS-like structures predominate on the biofilm and that these structures could be identified separated from organisms within the biofilm (Fig. 3B and 6). These membranous extensions are interesting. They can reach 10 to 15 μm in length (Fig. 3A and B). We presume that they are derived from the bacterial outer membrane, which is shed from the gonococcus in the form of blebs. Our studies have shown that human primary genital tract epithelial cells can serve as surfaces for gonococcal biofilm formation with minimal, if any, effect on cell viability. This may reflect the ability of the gonococcus to induce antiapoptotic factors in these cells (2, 3).
Immunoelectron microscopy experiments with primary urethral epithelial cells show membrane structures resembling those seen in Fig. 3A. These structures stained heavily with monoclonal antibody 6B4. It is interesting to speculate that these membranous structures are derived in large part from the fusions of blebs shed by the gonococcus and the meningococcus during growth (6). These may encase the gonococci within sac-like structures into which a matrix is released. A recent study of biofilms produced by 39 Neisseria meningitidis strains showed that 30% of the carriage isolates and 12.5% of the invasive disease isolates formed biofilms in microtiter wells (25). Generally, the more hydrophobic the surface of the organism, the more likely it was to form a biofilm, and encapsulation inhibited biofilm formation. These studies support the concept that hydrophobic interactions of surfaces with pathogenic Neisseria spp. enhance their ability to form biofilms. The present study lends support to the theory that the membranous bacterially derived structures may facilitate biofilm formation.
A number of previous studies have demonstrated that gonococci can persist in an asymptomatic state in the female genital tract (13). In addition, antibiotic resistance to a broad range of agents has become a major concern with the management of gonococcal infections (24). The ability of this organism to form a biofilm on human cells, particularly in the female genital tract, may be a factor in both of these consequences of gonococcal infection. Future studies will be directed at studying the nature of the biofilm matrix and the role of biofilms during infection of cervical epithelial cells.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant AI AI45728 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (to M.A.A).
We acknowledge the staff of the Central Microscopy Research Facility at the University of Iowa.
L.L.G. and J.L.E. contributed equally to the work described in this paper.
Present address: Columbus Children's Research Institute, Columbus, OH 43205.
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