bioﬁlm formation by neisseria gonorrhoeae · neisseria gonorrhoeae is a human-adapted,...

INFECTION AND IMMUNITY, Apr. 2005, p. 1964–1970 Vol. 73, No. 40019-9567/05/$08.00�0 doi:10.1128/IAI.73.4.1964–1970.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biofilm Formation by Neisseria gonorrhoeaeL. L. Greiner,† J. L. Edwards,†‡ J. Shao, C. Rabinak,

D. Entz, and M. A. Apicella*Department of Microbiology, University of Iowa, Iowa City, Iowa

Received 30 September 2004/Returned for modification 10 November 2004/Accepted 30 November 2004

Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could forma biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10�M sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with waterchannels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growthconditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmedthat 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 andabsence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and thelectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in thebiofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac andsialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primaryurethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Ourstudies 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.

Neisseria gonorrhoeae is a human-adapted, gram-negativediplococcus that infects the human male and female reproduc-tive tracts. N. gonorrhoeae infections in women frequently gounnoticed. This can eventually lead to serious upper genitaltract infections which ultimately can lead to infertility (13).Currently, no studies have discussed the ability of N. gonor-rhoeae to produce biofilms. Bacterial biofilms have been de-fined as communities of bacteria intimately associated witheach other and included within an exopolymer matrix. Thesebiological units exhibit their own properties, which are quitedifferent from those shown by the single species in planktonicform (15). Numerous bacterial species are capable of produc-ing biofilms. Biofilms confer a number of survival advantagesto the bacteria, including increased resistance to antimicrobialagents (7, 18).

Our interest in the capability of N. gonorrhoeae to form abiofilm came about by observations made in our laboratoryduring 4- and 8-day infections of primary human urethral andcervical epithelial cells (8, 12). Those studies showed that thegonococcus was forming microcolonies on these surfaces, andeventually these transitioned into structures that resembledbacterial biofilms.

The purpose of this study was twofold. The first objectivewas to verify that N. gonorrhoeae can produce a biofilm both inbiofilm chambers and over primary human genital tract epi-thelial cells in culture. The second objective was to gain infor-

mation about the structure of the gonococcal biofilm. To ac-complish this, we established gonococcal infections incontinuous-flow chambers and on primary human genital tractepithelial cells. Light and electron microscopic analyses indi-cated that the gonococcus could form a biofilm on these sur-faces and that the ultrastructure resembled that previouslyseen with other bacteria, with the exception that the biofilmwas interlaced with what appeared to be membranous struc-tures surrounding the organisms. Lectin and antibody analysesindicated that the biofilm sugars were similar to those thatwere expressed as terminal saccharides of gonococcal lipooli-gosaccharide (LOS).

MATERIALS AND METHODS

Bacteria and culture conditions. N. gonorrhoeae strains 1291, FA1090, andMS11, used in this study, are clinical isolates. These strains were reconstitutedfrom frozen stock cultures and propagated at 37°C with 5% CO2 on GC agar(Difco, Detroit, Mich.) supplemented with 10 ml of IsoVitaleX (Becton-Dick-inson, Franklin Lakes, N.J.) per liter. These N. gonorrhoeae strains also weretransformed with pGFP (pLES98 containing gfp) to express green fluorescentprotein (GFP). The plasmid pLES98 was a gift from V. Clark. The strain numberfollowed by pGFP in brackets is used to designate these transformed strains.

Biofilm growth in a continuous-flow chamber. Gonococcal strains were grownin a continuous-flow chamber identical to that described by Davies et al. (5). RPMI1640 medium (Gibco, Grand Island, N.Y.) containing 100 �M sodium pyruvate(Gibco), 20 ml of hypoxanthine (370 �M) and uracil (450 �M) per liter, 100 �Msodium nitrite, and 1% IsoVitaleX was prepared. In some experiments, 20 �MCMP-N-acetylneuraminic acid (CMP-Neu5Ac) was added to the medium. Thissolution was diluted 1:10 with sterile phosphate-buffered saline (PBS) and was usedin the continuous-flow experiments. To inoculate the chamber, 1 ml of N. gonor-rhoeae culture, grown to a density of 108 organisms/ml, was placed in the chamberand left for 1 h. The flow was then started at 150 �l/min. The biofilm was formed ina 37°C environmental incubator and continuously perfused over the duration of theexperiment. At the end of that time period, the effluent was cultured to assure thatthe culture purity was maintained. Digital biofilm images were then collected.

Laser scanning confocal microscopy of continuous-flow chambers and cul-tured epithelial cells. Confocal images of biofilms in continuous-flow chambersand on primary human cervical epithelial cells in culture were obtained with a

* Corresponding author. Mailing address: Department of Microbi-ology, University of Iowa, 51 Newton Rd., Iowa City, IA 52242. Phone:(319) 335-7807. Fax: (319) 335-9006. E-mail: [email protected].

† L.L.G. and J.L.E. contributed equally to the work described in thispaper.

‡ Present address: Columbus Children’s Research Institute, Colum-bus, OH 43205.

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Bio-Rad MRC-1024 scanning confocal microscope as previously described (8).The chambers were viewed in situ under the confocal microscope. Cervicalepithelial cells were grown on collagen-coated glass coverslips in a 24-well plate.Infections were performed in the chambers, and at various time points thecoverslips were removed from the chambers and viewed with the confocal mi-croscope after appropriate staining.

Viability staining of bacteria from continuous-flow chamber biofilms. Toevaluate the viability of bacteria present within the biofilm matrix, N. gonorrhoeae1291 was grown in a flow chamber as described above. After 4 days, the flowchamber was carefully disconnected. The Live/Dead BacLight bacterial viabilitykit (Molecular Probes, Eugene, Oreg.) was used to visualize live and deadbacteria within the biofilm. Briefly, SYTO 9 (component A) and propidiumiodide (component B) were mixed at a 1:1 ratio. Three microliters of the viabilitystain was added to 1 ml of PBS. Medium in the chamber was aseptically replacedwith the stain-PBS mixture. The chamber was incubated for 15 min at 37°C. Onemilliliter 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 510laser scanning confocal microscope at a magnification of �10. The resultingimages were compiled as cross-sections of a z series.

Fixation of biofilm samples for microscopy. All samples used for microscopywere grown in biofilm chambers in RPMI 1640 medium as described above. In allexperiments other than the live/dead studies, a 1-ml mixture of 4% paraformal-dehyde and 5% dimethyl sulfoxide was infused into the chamber at the end of thegrowth period and allowed to fix overnight. Samples were then embedded in situin OCT resin (Sakura Finetek USA, Inc., Torrance, Calif.) on the coverslipsurface upon which they were formed. After hardening, the coverslip was re-moved by freezing the sample in liquid nitrogen and shattering the glass, leavingthe biofilm within the OCT resin. The biofilm was then cut into 1-�m-thicksections. N. gonorrhoeae strain 1291 was used for microscopic analyses unlessotherwise noted.

Microscopy. OCT cryosections were incubated with hematoxylin and eosinstains to visualize the biofilm matrix. Images were viewed with an Olympus lightmicroscope.

Biofilm samples from continuous-flow chambers were prepared for transmis-sion electron microscopy (TEM) by using perfluorocarbon methods to minimizethe 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-kVaccelerating voltage as previously described (12).

Biofilms grown on primary human cervical epithelial cells were processed forscanning electron microscopy (SEM) and viewed with a Hitachi S-4000 scanningelectron 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. Pro-cessed coverslips were then mounted onto stubs with colloidal silver and weresputter coated with gold palladium.

Biofilm samples grown in the presence of CMP-Neu5Ac were cultured (withgentle agitation) on glass coverslips, fixed with 2.5% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.2), and rinsed in double-distilled H2O immediatelybefore plunge-freezing in liquid nitrogen. Frozen specimens were introducedinto an Emitech K1250 cryogenic system preparation chamber for radiant heatsurface 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 HitachiS-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 ofIowa Central Microscopy Research Facility (Iowa City).

Lectin analysis of biofilms. The OCT resin sections were studied by fluores-cence microscopy with the fluorescein-conjugated lectins Maackia amurensis,Sambucus nigra, succinylated wheat germ, soybean agglutinin, and Amaryllis (allfrom EY Laboratories, San Mateo, Calif.) as previously described (10).

Antibody analysis of biofilms. OCT embedded sections were incubated withmonoclonal antibodies (MAbs) 6B4 (murine immunoglobulin M [IgM]) and 2C3(murine IgG). Anti-murine IgM–fluorescein isothiocyanate and anti-IgG–tetra-methyl rhodamine isocyanate were used as secondary antibodies. Images of thesamples were taken with the Bio-Rad MRC-1024 laser scanning confocal viewingsystem.

Gonococcal biofilm formation on primary human cervical epithelial cells.Surgical biopsies derived from the ecto- and the endocervix that were used toseed primary cervical epithelial cell systems were procured and maintained asdescribed previously (5) in defined keratinocyte serum-free medium (Life Tech-nologies, Rockville, Md.). Primary cervical epithelial cells were grown on cover-slips 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 of100. 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 exper-iments, samples were infected with N. gonorrhoeae 1291[pGFP], fixed, and thenstained with ethidium bromide. These samples were viewed with the Bio-RadMRC-1024 laser scanning confocal viewing system.

RESULTS

Continuous-flow chamber studies. To test the hypothesisthat gonococci could form biofilms, we performed studies inwhich continuous-flow chambers (7) were infected for 1 h with108 piliated strain 1291 gonococci. At the end of that timeperiod, the chamber was continually perfused at a flow rate of150 �l/min with supplemented RPMI 1640 containing 10 �Msodium nitrite. Nitrite was initially added to ensure gonococcalsurvival in the relatively low-oxygen environment of the flowcell. Figure 1A shows the result of a typical 5-day flow chamberstudy with N. gonorrhoeae strain 1291. As can be seen, approx-imately 80% of the coverslip surface is covered with whatappears to be a biofilm. Flow eddies can be seen where thebiofilm had been washed off the coverslip by the flowing me-dium. Attempts to reduce the flow rate in the chamber resulted

FIG. 1. Panel A shows a typical 4-day biofilm formed in the con-tinuous-flow chamber by N. gonorrhoeae, and panel B shows the resultsof confocal analysis of a vertical reconstruction of a z series usinglive/dead staining of the biofilm. The majority of the organisms nearestthe stream of flow are viable. Panels C and E show confocal analysis ofstrain 1291[pGFP] 4-day biofilm produced in defined medium in thepresence of 10 �M sodium nitrite. The bars in these panels indicate 20�m. Panel C shows a horizontal three-dimensional reconstruction of 60images at 1-�m intervals (a stacked z series), and panel E shows thevertical view of the same stacked z series. Panels D and F show confocalanalysis of strain 1291[pGFP] 4-day biofilm produced in defined mediumin the absence of sodium nitrite. Panel D shows the stacked z series, andpanel F shows the vertical view of the same stacked z series. (C to F) Bar,20 �m.

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in increased biofilm formation and obstruction of the chamberflow. We performed live/dead staining immediately after thetermination of the experiment (Fig. 1B). The clusters of or-ganisms nearest the surface of the glass coverslip appeared tobe nonviable, while those closer to the medium flow streamappeared to be viable. Gonococcal strains MS11 and FA1090were also studied in the flow chamber and were found to becapable of forming biofilms (data not shown). We assumedthat within the chamber the gonococcus would need nitrite asan electron acceptor, as we believed that the chamber wouldconstitute a microaerophilic environment. Our studies indi-cated that the gonococcus would grow in the flow chamber inthe absence of nitrite. Figures 1C and E and 1D and F showconfocal analyses of a biofilm produced by N. gonorrhoeaestrain 1291 in the continuous-flow chamber over 4 days in thepresence (final concentration, 10 �M) and absence of nitrite,respectively. These studies suggested that the biofilm topogra-phy might vary under these different conditions. Biofilm for-mation in the absence of nitrite suggested that the chamberenvironment was not microaerophilic or, alternatively, thatother factors such as cytochrome oxidase might facilitate cellgrowth under microaerophilic conditions.

Microscopic analyses. We performed initial light microscopystudies of stained cryosections of biofilms taken from cover-slips. To maintain the in situ conformation of the biofilm, thesamples were cryopreserved in dimethyl sulfoxide, embeddedin OCT, snap frozen, and sectioned on a cryomicrotome. Ascan be seen in Fig. 2A, in a hematoxylin- and eosin-stainedsection, the biofilm had numerous water channels. In addition,the organisms within the biofilm were surrounded by whatappears to be a pink-staining matrix (Fig. 2A).

We performed C-FESEM on a 3-day N. gonorrhoeae 1291biofilm. Figure 2B and C and shows organisms embedded in acontinuous matrix. Demarcated water channels could beclearly seen within the biofilm. In addition, what appeared tobe membrane-like structures spanned areas of the biofilm (Fig.2C). Transmission electron microscopy of biofilm samplesfixed in perfluorocarbon also revealed extensive membrane-like structures enclosing organisms within the biofilm, withtraces of what appeared to be residual matrix material (Fig. 3Aand B). TEM analysis of the apical portion of the biofilmshowed a mass of membrane-like structures covering organ-isms (Fig. 3C). Figure 3D shows an immunoelectron micro-graph of a gonococcal biofilm on a primary human urethralepithelial cell. Similar membrane-like structures were seenwithin these biofilms. These membranes strained with mono-clonal antibody 6B4, which recognizes a lactosamine epitopeknown to be present on gonococcal LOS (1).

Lectin analysis of the biofilm. We also performed lectin-binding studies with frozen, OCT-embedded biofilm formed inthe absence of CMP-Neu5Ac (Fig. 4). Sections were labeledwith the following fluorescein-conjugated lectins: succinylatedwheat germ agglutinin (primary specificity, N-acetylglu-cosamine), S. nigra (primary specificity, Neu5Ac �2-6Gal), M.amurensis (primary specificity, Neu5Ac �3-6Gal), Amaryllis(primary specificity, mannose), and soybean agglutinin (pri-mary specificity, �-linked N-acetylgalactosamine; secondaryspecificity, galactose). The only lectin that bound to the biofilmwas soybean agglutinin. This suggested that the terminal sugaron the biofilm matrix when grown in the absence of CMP-

Neu5Ac was either a �-linked N-acetylgalactosamine or a ga-lactose. To test whether Neu5Ac is incorporated into the biofilm,CMP-Neu5Ac (10 �M) was added to the growth medium. Lectinanalysis demonstrated that soybean agglutinin failed to bind tothe biofilm formed in the presence of CMP-Neu5Ac and thatbinding was restored after neuraminidase treatment of the biofilm(Fig. 5). M. amurensis lectin bound to the sialylated biofilm, andthis binding was eliminated by treatment with neuraminidase.These studies indicated that NeuAc could be incorporated intothe biofilm if the substrate CMP-Neu5Ac was available.

Antibody analysis of the biofilm. To confirm the lectin stud-ies, biofilm embedded in OCT was incubated with MAbs 6B4(murine IgM) and 2C3 (murine IgG). Monoclonal antibody6B4 recognizes a Gal�1-4GlcNAc epitope (which is presentalso on strain 1291 LOS), and MAb 2C3 binds to the H.8protein of pathogenic Neisseria spp. Anti-murine IgM–fluores-cein isothiocyanate and anti-IgG–tetramethyl rhodamine iso-cyanate were used as secondary antibodies. If the biofilm hada terminal lactosamine structure (Gal�1-4GlcNAc), we pre-dicted that we might detect green fluorescence surroundingyellow-orange-staining organisms. Figure 6 shows the results ofthis experiment, which demonstrates this and indicates that thebiofilm matrix itself binds MAb 6B4, as areas in which 6B4binding occurs without colocalization with 2C3-labeled bacte-ria can be seen. Similar studies were performed on biofilmformed in the presence of CMP-Neu5Ac. These studies dem-onstrated that MAb 6B4 failed to bind the sialylated biofilmbut that after treatment with neuraminidase, MAb 6B4 didbind to the biofilm (data not shown). Thus, the conclusionsfrom the lectin and monoclonal antibody studies indicated thatpredominate terminal sugars of the biofilm in the absence andpresence of CMP-Neu5Ac are lactosamine and sialyllac-tosamine, respectively.

Gonococcal biofilm formation on primary human cervicalepithelial cells. Our hypothesis that gonococci produced bio-films originated when we observed microcolonies and whatappeared to be biofilms on cultured primary human genitaltract cells after 3- and 4-day infections. Figure 7 shows a con-focal study of human primary cervical epithelial cells infectedwith N. gonorrhoeae strain 1291[pGFP]. A biofilm layer extend-ing approximately 20 to 30 �m above the primary humancervical epithelial cells in what appeared to be tower-like for-mations was seen. The epithelial cell layer remained intactduring these experiments, and trypan blue exclusion studiesdemonstrated that over 95% of the infected epithelial cellswere viable at the termination of the experiment at 4 and 8days. SEM analysis of 4- and 8-day gonococcal infections re-vealed biofilm formation over cervical epithelial cells that cov-ered almost the entire epithelial cell surface by day 8 afterinitiation of infection (Fig. 8).

DISCUSSION

These studies demonstrated that N. gonorrhoeae could forma biofilm in a continuous-flow chamber and over primary hu-man genital tract epithelial cells in culture. Biofilms are com-plex communities of bacteria that develop in diverse environ-ments (11). They are dynamic structures in which cells switchfrom a weak interaction with the surface to an almost perma-nent binding through extracellular polymers. In addition, the

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bacteria form channels and pores to provide nutrients to bac-teria within the biofilm structure. The maintenance of a biofilmis attributed to the development and preservation of the ex-opolysaccharide matrix (4). More than 300 proteins that arenot detected in planktonic bacteria can be detected in bacteriafrom mature biofilms (20). These proteins fall into the func-tional classes of metabolism, phospholipid and lipopolysaccha-ride biosynthesis, membrane transport and secretion, and ad-aptation and protective mechanisms. In addition, biofilmbacteria are considered to be in the stationary phase of growth,partly because of the accumulation of acylhomoserine lactonewithin cell clusters (23). Some bacteria eventually detach fromthe mature biofilm to enter the surrounding fluid phase. De-

tachment is a physiologically regulated event in which bacteriawill release from the biofilm as a planktonic organism to moveon to attach to other surfaces, where they initiate biofilm de-velopment (16). Many different mechanisms may contribute tothe detachment process. O’Toole and Kolter (17) demon-strated that starvation may lead to detachment by an unknownmechanism. 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 en-zyme could be cell density. In addition, the presence of homo-serine lactones may cause biofilm reduction, as has been dem-onstrated with Rhodobacter sphaeroides (17, 19).

These studies have utilized several microscopic approaches

FIG. 2. Panel A shows a hematoxylin- and eosin-stained section of gonococcal strain 1291 4-day biofilm embedded in OCT and sectioned ona cryomicrotome. This panel shows organisms (dark blue) surrounded by a staining pink matrix. Panels B, C, and D show C-FESEM images ofa 3-day biofilm at different magnifications. Panel B, taken at a magnification of �1,000, shows a broad view of the surface. Arrows point to anoverlying membrane covering the biofilm. Panel C (magnification, �10,000) shows evidence of organisms embedded within membranous structuresthat appear to contain a matrix. Panel D (magnification, �20,000) demonstrates typical membranous structures seen crossing the biofilm.



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to elucidate the structure of the biofilm produced by N. gon-orrhoeae. Cryosections viewed by light microscopy indicatedthat organisms were surrounded by a matrix (Fig. 2A).Cryofield emission scanning electron microscopy and TEMhave shown that this biofilm consists of membranous structuresthat appeared to be surrounding gonococci that may also beencased in a matrix. Binding studies with lectins and monoclo-nal antibody 6B4 showed that LOS-like structures predomi-nate on the biofilm and that these structures could be identi-fied separated from organisms within the biofilm (Fig. 3B and

6). These membranous extensions are interesting. They canreach 10 to 15 �m in length (Fig. 3A and B). We presume thatthey are derived from the bacterial outer membrane, which isshed from the gonococcus in the form of blebs. Our studieshave shown that human primary genital tract epithelial cellscan serve as surfaces for gonococcal biofilm formation withminimal, if any, effect on cell viability. This may reflect theability of the gonococcus to induce antiapoptotic factors inthese cells (2, 3).

Immunoelectron microscopy experiments with primary ure-

FIG. 3. Transmission electron micrographs of perfluorocarbon-fixed 3-day biofilm embedded in Epon resin. Panel A shows sections from the surfaceof the biofilm closest to the glass coverslip. Organisms can be seen contained within membranes. Panel B shows sections from the middle portion of thebiofilm, showing organisms, membranes (arrows), and residual matrix. Panel C shows the top of the biofilm covered with membranes (dotted arrow).Panel D shows a immunoelectron micrographic study of a 3-day infection of primary human urethral epithelial cells (UEC). The section was incubatedwith MAb 6B4 as the primary antibody and with a goat anti-murine IgM gold conjugate as the secondary antibody. Membranous structures could befound over the epithelial cell surface surrounded by organisms. These membranes were covered with colloidal gold particles.



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thral epithelial cells show membrane structures resemblingthose seen in Fig. 3A. These structures stained heavily withmonoclonal antibody 6B4. It is interesting to speculate thatthese membranous structures are derived in large part fromthe fusions of blebs shed by the gonococcus and the meningo-coccus during growth (6). These may encase the gonococciwithin sac-like structures into which a matrix is released. A

recent study of biofilms produced by 39 Neisseria meningitidisstrains showed that 30% of the carriage isolates and 12.5% ofthe invasive disease isolates formed biofilms in microtiter wells(25). Generally, the more hydrophobic the surface of the or-

FIG. 4. Lectin staining of a 4-day gonococcal strain 1291 biofilm. Thebiofilm was cyropreserved, embedded in OCT, and cyrosectioned. All ofthe lectins are conjugated to fluorescein. Panel A shows unstained biofilm.The remaining panels were stained with S. nigra (B), succinylated wheatgerm (C), M. amurensis (D), soybean agglutinin (E), and Amaryllis (F).These studies suggest that the terminal sugar in the biofilm matrix is eithera �-linked-N-acetylgalactosamine or a galactose.

FIG. 5. Lectin staining of an N. gonorrhoeae 1291 3-day biofilmgrown in the presence of CMP-Neu5Ac. Panel A shows staining by M.amurensis before treatment with 0.05 U of neuraminidase/ml, andpanel B shows a lack of binding after treatment with neuraminidase.Panel C shows a lack of binding of soybean agglutinin lectin beforetreatment with neuraminidase, and panel D shows restoration of bind-ing after neuramindase treatment. This study indicates that structureswithin the biofilm can be sialylated in the presence of CMP-Neu5Ac.

FIG. 6. Confocal microscopy of cyrosectioned 4-day gonococcalstrain 1291 biofilm incubated with MAb 2C3 (red channel, panel A)and MAb 6B4 (green channel, panel B). MAb 2C3 is specific for anH.8 protein of pathogenic Neisseria spp., and MAb 6B4 is specific forthe Gal�1-4GlcNAc epitope. Panel C shows the merged image. Thesolid arrows designate the organisms in the biofilm in which MAbs 6B4and 2C3 colocalize. The dotted arrows designate the regions of thebiofilm binding MAb 6B4 and not colocalizing with strain 1291.

FIG. 7. Tilted stacked z series of a confocal analysis of N. gonor-rhoeae strain 1291 infected for 4 days over primary human cervicalepithelial cells. The tissue culture medium was changed daily. Thebiofilm rises 20 to 30 �m above the cell layer. This tilted image of the1291-infected sample demonstrates the topography of the biofilm overthe epithelial cell surface at 4 days.



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ganism, the more likely it was to form a biofilm, and encapsu-lation inhibited biofilm formation. These studies support theconcept that hydrophobic interactions of surfaces with patho-genic Neisseria spp. enhance their ability to form biofilms. Thepresent study lends support to the theory that the membranousbacterially derived structures may facilitate biofilm formation.

A number of previous studies have demonstrated that gono-cocci can persist in an asymptomatic state in the female genitaltract (13). In addition, antibiotic resistance to a broad range ofagents has become a major concern with the management ofgonococcal infections (24). The ability of this organism to forma biofilm on human cells, particularly in the female genitaltract, may be a factor in both of these consequences of gono-coccal infection. Future studies will be directed at studying thenature of the biofilm matrix and the role of biofilms duringinfection of cervical epithelial cells.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant AI AI45728from the National Institute of Allergy and Infectious Diseases, Na-tional Institutes of Health (to M.A.A).

We acknowledge the staff of the Central Microscopy Research Fa-cility at the University of Iowa.

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Editor: T. R. Kozel

FIG. 8. SEM of uninfected cervical cells (panel A) and of a 4-dayinfection of primary human cervical epithelial cells with strain 1291(panels B and C). Panel D shows the results of an 8-day infection. Thedotted arrow in panel B designates the cervical epithelial cell surface.The retracted matrix can be seen covering the top of the structure(solid arrow, panel B). The epithelial cell surface can also be seen(dotted arrow). Panel C shows a high-magnification (�25,000) view ofthe matrix covering the organisms (arrow). Panel D shows almost theentire epithelial cell surface covered by a biofilm at 8 days.



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