Biofilm Formation and Interaction with the Surfaces of Gallstones by Salmonella spp.

Salmonellae can exist in an asymptomatic carrier state in thehuman gallbladder. Individuals with gallstones are more likelyto become typhoid carriers, and antibiotic treatments are oftenineffectual against Salmonella enterica serovar Typhi in carrierswith gallstones. Therefore, we hypothesized that Salmonellaspp. form biofilms on the surfaces of gallstones, where thebacteria are protected from high concentrations of bile andantibiotics. A number of methods were utilized to examine biofilmformation on human gallstones and glass coverslips in vitro,including confocal, light, and scanning electron microscopy.In our assays, salmonellae formed full biofilms on the surfacesof gallstones within 14 days and appeared to excrete an exopolysaccharidelayer that bound them to the surfaces and to other bacteria.Efficient biofilm formation on gallstones was dependent uponthe presence of bile, as a biofilm did not form on gallstoneswithin 14 days in Luria-Bertani broth alone. The biofilms formedby a Salmonella enterica serovar Typhi Vi antigen mutant, aswell as strains with mutations in genes that eliminate productionof four different fimbriae, were indistinguishable from thebiofilms formed by the parents. Mutants with an incomplete O-antigen,mutants that were nonmotile, and mutants deficient in quorumsensing were unable to develop complete biofilms. In addition,there appeared to be selectivity in salmonella binding to thegallstone surface that did not depend on the topology or surfacearchitecture. These studies should aid in the understandingof the Salmonella carrier state, an important but underresearchedarea of typhoid fever pathogenesis. If the basis of carrierdevelopment can be understood, it may be possible to identifyeffective strategies to prevent or treat this chronic infection.

INTRODUCTION

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Introduction

Biofilms have recently been implicated as the cause of manychronic infections in humans and are frequently associated withimplanted devices, such as catheters, prosthetics, and contactlenses (for reviews see references 10 and 12). A biofilm isdefined as a population of one or more organisms attached toeach other and a surface by means of a bacterium-initiated matrix(10, 12). This matrix provides a very stable environment andresults in high levels of resistance to antimicrobial agents.Often, it is difficult to clear the infection unless the substrateto which the bacteria are attached is removed. From the sessile,matrix-bound community, planktonic cells can be continuouslyshed from the biofilm, which in humans can lead to a systemicinfection or release of the organism into the environment.

Salmonella enterica serovar Typhi, which causes typhoid feverin humans, crosses the intestinal epithelial layer and invadesmacrophages. The macrophages carry the organism to the liver,pancreas, and spleen, where the bacteria are thought to replicatein both phagocytic and nonphagocytic cells. From the liver,the bacteria can be shed into the gallbladder, where eitheran active infection (cholecystitis) or a chronic infection (carrierstate) can develop. The carrier state occurs in about 3 to 5%of the people infected and is frequently associated with gallbladderabnormalities, such as gallstones (15, 20). This infection isoften asymptomatic and can last for many years with little orno deleterious effect on the host. Furthermore, it has beenshown that the chronic carrier state is the single most importantrisk factor for development of hepatobiliary carcinomas, asevidenced by the finding that salmonella carriers with gallstoneshave an 8.47-fold-higher risk for developing cancer of the gallbladder(15, 33).

It is interesting that the gallbladder is the primary site ofcarriage, considering that it is also the storage site for bile.Bile is produced in the liver and consists of many components,including bile salts, cholesterol, and bilirubin. Bile saltsare detergents that aid in degradation and dispersion of lipidsand as such make bile a good antimicrobial agent. Salmonella,however, is resistant to high concentrations of bile and individualbile salts (38). In addition to being highly resistant to theeffects of bile, the levels of several proteins of Salmonellaspp. are affected both positively and negatively by bile (38).Bile has also been shown to be important as an environmentalsignal for Salmonella invasion of epithelial cells (28). Thepresence of bile leads to down-regulation of the transcriptionof genes in the type III secretion system of pathogenicity islandI. This leads to a reduction in the amount of effector moleculessecreted and reduced invasion of epithelial cells. Therefore,bile appears to be an important environmental signal for Salmonellapathogenesis (as well as pathogenesis with other enteric pathogens[17]), probably during both acute and chronic infections.

Antibiotic treatment can be ineffective in Salmonella carrierswith gallstones, and elimination of gallbladder infection inthese individuals usually requires surgery and gallstone removal(20). However, this option is less likely in several developingcountries where S. enterica serovar Typhi is prevalent. Thus,it is important to determine the mechanisms by which Salmonellacan establish gallbladder carriage and, possibly most importantly,to determine how it does so in the presence of gallstones. BecauseSalmonella spp. cause chronic infections of the gallbladderand gallstones are a major factor influencing the formationof the carrier state, in this study we addressed the hypothesisthat salmonellae form biofilms on the surfaces of gallstones.Such biofilm formation may contribute to establishment of thechronic carrier state. In addition, the roles of bile and variousbacterial surface structures in Salmonella biofilm formationon gallstones were examined.

MATERIALS AND METHODS

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Materials and Methods

Strains and plasmids. The Salmonella strains and plasmid used in this study are listedin Table 1. Plasmid pFPV25.1 was transformed into ATCC 14028sand Ty2 by electroporation, and Luria-Bertani (LB) agar platescontaining ampicillin (50 µg/ml) were used to select forthe plasmid.

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TABLE 1. Bacterial strains, plasmid, and relevant characteristics

Growth conditions. One group of uniform gallstones composed mainly of cholesteroland removed from a single patient was used in these studies.Human gallstones were incubated in test tubes with a strainof Salmonella in either LB broth or LB broth with 3% ox bile(Sigma, St. Louis, Mo.) with aeration at 37°C. Every 24h, the medium was removed, the gallstones were washed two timesin LB broth by vigorous vortexing, and fresh medium was added.Antibiotics (50 µg of ampicillin per ml, 25 µg ofchloramphenicol per ml, 15 or 10 µg of tetracycline perml, and 45 µg of kanamycin per ml) were added where appropriate.The growth periods varied from 4 to 18 days as indicated below.

Glass coverslips were placed in the bottom of a petri dish andcovered with 10 ml of medium. The medium used for the coverslipswas tryptic soy broth (TSB) or LB broth either with or without3% ox bile. Strains were inoculated into the plate by using1:100 dilutions of overnight cultures. Antibiotics were added,and the plates were rocked slowly overnight at 37°C. Every24 h the medium was removed, each preparation was washed twotimes in 1x phosphate-buffered saline (PBS), and fresh mediumwith antibiotics was added. Enough glass slides were placedinto the petri dishes so that one could be removed every dayfor 14 days.

Crystal violet staining of biofilms on glass slides. Removed glass slides were washed with a continuous spray of1x PBS and incubated at 60°C for 1 h to fix the cells. Then0.1% crystal violet (gentian violet in isopropanol-methanol-1xPBS [1:1:18]) was placed on each glass slide, and the slideswere incubated for 15 min at room temperature. The slides werethen washed thoroughly with 1x PBS until the PBS ran clear.The slides were then broken, put in Eppendorf tubes, and immersedin 33% acetic acid to extract the dye. The optical density at570 nm of the acetic acid solution was assessed to determinethe amount of dye retained by the bacterial cells.

Ruthenium red staining of glass slides for detection of EPS. Glass slides were incubated in petri dishes as described above,removed, and stained as described by Mills et al. (23). Briefly,a slide was placed in a 12-well cell culture cluster (Corning,Corning, N.Y.) with 1 ml of 0.15% ruthenium red-0.5% glutaraldehydedissolved in 100 mM cacodylate buffer for 1 h at room temperature(Sigma). This stain was removed, 0.05% ruthenium red-5% glutaraldehydein a 100 mM cacodylate buffer solution was added, and the preparationwas incubated for 2 h at room temperature. This stain was removed,and the slides were washed five times for 10 min in 100 mM cacodylatebuffer. The slides were then ready to be viewed by light microscopyfor exopolysaccharide (EPS) staining.

Confocal scanning laser microscopy (CSLM). After growth for a selected period of time, gallstones werewashed two times in LB broth with vortexing and placed in aNunc Labtek chambered coverslip for immediate viewing. Strainsconstitutively producing green fluorescent protein were scannedwith a Fluoview Scanhead using an argon ion laser with excitationat 488 nm. The biofilm was viewed with a x40 dry objective usinga 1X70 Olympus microscope, and images were viewed by using Fluoviewsoftware, version 2.1.

Scanning electron microscopy (SEM). After samples were grown for a specified period of time, gallstoneswere washed two times with vortexing and placed into 2% glutaraldehydeto fix the specimens. The specimens were then dehydrated ingraded ethanol and Freon (FC-113) series and dried in an OmarSPC1500 critical point dryer. The specimens were then sputtercoated with a Technics Hummer V and viewed with a JEOL JSM-840Ascanning electron microscope.

RESULTS

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Results

Salmonella spp. form biofilms on the surfaces of gallstones To determine if Salmonella spp. can form biofilms in vitro ongallstones, human gallstones were exposed to Salmonella spp.in liquid cultures with aeration at 37°C. The medium wasreplaced every 24 h in an attempt to mimic gallbladder emptying.After the specified time, the gallstones were examined for thepresence of biofilms either by CSLM or by SEM. To examine gallstonesfor biofilm formation by CSLM, strains of S. enterica serovarTyphimurium (ATCC 14028s) and S. enterica serovar Typhi (Ty2)constitutively producing green fluorescent protein were used.Gallstones were exposed to the bacteria for 7, 11, or 18 daysin LB broth containing 3% bile prior to examination by microscopy.Both S. enterica serovar Typhimurium and S. enterica serovarTyphi were bound to most of the gallstone surface after 11 days(but not after 7 days), and there was nearly full coverage ofthe gallstones after 18 days (Fig. 1A). Each biofilm was alsoexamined for depth by assembly of a three-dimensional imagefrom CSLM Z-scans. The data demonstrated that the depth of thebiofilm was significant ( $~$ 31 µm), and because salmonellaeare $~$ 2 µm in length, the cells clearly were in a multilayercommunity (Fig. 1B). In addition, visual inspection of the Z-scansrevealed evidence of water channels, which are typical in fullyformed biofilms.

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FIG. 1. CSLM micrographs of gallstones incubated with S. enterica serovar Typhi constitutively producing green fluorescent protein. (A) S. enterica serovar Typhi cells exposed to a gallstone for 18 days in the presence of 3% bile in LB broth are distributed over most of the surface of the gallstone. However, note the pockets of the stone surface where no bacteria appear to bind, suggesting that there is specificity in the Salmonella-gallstone interaction. Bar = 10 µm. (B) Images taken through the depth of the biofilm using a Z-scan were assembled into a three-dimensional form. The micrograph represents a thickness of $~$ 31 µm, and because salmonellae are $~$ 2 µm long, the results demonstrate that the cells do not exist as a monolayer.

Gallstones exposed to wild-type S. enterica serovar Typhimuriumand S. enterica serovar Typhi were also examined by SEM forbiofilm formation, as well as for biofilm characteristics. Similarto what was observed by CSLM, both S. enterica serovar Typhimuriumand S. enterica serovar Typhi were found to completely coverthe surfaces of the gallstones by 14 days (Fig. 2B). The imagesdemonstrated that the bacteria did not form densely packed monolayersbut rather formed loosely assembled matrices of cells, whichare typical of biofilms formed by other species on various surfaces.In addition to material that appeared to be typical of a bacterialEPS in both Salmonella serovars, a web-like network of strandsthat connected the bacteria to one another and to the gallstonesurface was observed (Fig. 2C). Previous reports suggested thatsimilar structures may result from the condensation of EPS thatoccurs during the fixation process (7). However, it is alsopossible that these strands are unique to Salmonella gallstonebiofilms. Therefore, both CSLM and SEM demonstrated that Salmonellaspp. can form biofilms on gallstone surfaces in vitro but thatthis process was rather slow in our culture system comparedto formation of biofilms of other bacterial species, which candevelop in as little as a few hours or a few days.

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FIG. 2. SEM micrographs of S. enterica serovar Typhimurium biofilm formation on the surfaces of human gallstones. (A) Example of the gallstone surface on which Salmonella are most commonly found. The gallstone was grown in the presence of bacteria in LB broth alone, and the results demonstrate that salmonellae do not form a biofilm or adhere to the surface unless bile is present in the medium. (B) Wild-type Salmonella cells exposed to a human gallstone for 14 days in the presence of LB broth containing 3% bile are loosely dispersed over the surface of a gallstone with apparent desiccated EPS. (C) Increased magnification of panel B, showing the web-like strands connecting bacteria both to other bacteria and to the gallstone surface.

Biofilm formation is bile dependent. Once it was determined that biofilm formation occurs on gallstones,it was of interest to examine the conditions needed to forma biofilm. Because the gallbladder is the storage site for bile,it is hypothesized that bile may act as a signal for biofilmformation to occur. Therefore, gallstones were incubated inthe presence of either LB broth alone or LB broth containing3% bile. The presence of bile induced the formation of a fullbiofilm on a gallstone after 14 days in both S. enterica serovarTyphimurium and S. enterica serovar Typhi cultures, whereasno biofilm was observed after 14 days on the gallstones in LBbroth without bile (Fig. 2). Therefore, in our model system,Salmonella biofilm formation on gallstones was dependent uponbile in the culture medium.

Because bile is necessary for biofilm formation on gallstonesto occur in 14 days, it is likely that bile either signals thebacteria to form a biofilm or conditions the surfaces of thegallstones, making adhesion easier. To test this hypothesis,gallstones without bacteria were placed in either LB broth aloneor LB broth with 3% bile and incubated for 7 days. Bacteriawere then added, and the cultures were treated as describedabove for 4 days, fixed, and viewed by SEM. In addition, planktonicbacteria were grown in LB broth or LB broth with 3% bile at37°C for 7 days with the cultures back-diluted into freshmedium every 24 h. After the 7 days, the bacteria were incubatedwith a gallstone for 4 days, fixed, and viewed by SEM. The resultsshowed that neither incubating the bacteria nor conditioningthe gallstones with bile before the gallstones and bacteriawere incubated together induced full biofilm formation after4 days (data not shown). These results indicate that preconditioningbacteria or gallstones with bile is not sufficient to initiaterapid biofilm formation. Therefore, it is likely that the bacteriareceive signals both from bile and from interacting with thegallstone surface to initiate biofilm development.

Biofilm formation is surface specific. Bacterial biofilms have been found on a variety of biotic andabiotic surfaces (10, 12). To test the specificity of Salmonellabiofilm formation on gallstones, bacteria were incubated underidentical conditions (LB broth containing 3% bile) with eithera gallstone or a granite pebble that was a similar size. After14 days, the gallstone and the pebble were examined by SEM.Although some bacteria were found to be bound to the pebblesurface, the characteristics of the bound bacteria did not resemblethe characteristics of the bacteria in a biofilm on the gallstone.The bacteria, which were in clumps, were tightly compacted onthe surface of the pebble in scattered patches with little visibleEPS (data not shown). Furthermore, the number of bacteria boundto the gallstone was much larger than the number of bacteriabound to the pebble.

The surface of an individual gallstone is not uniform, eitherin composition or in topology. Examination of both SEM and CSLMimages showed that while salmonellae readily bound to the gallstonesurface, there were patches on the surface to which the bacteriadid not adhere (Fig. 1A). Based on the SEM studies, surfacespecificity does not appear to be due to vast differences ingallstone topology, as an unbound surface can be observed inthe same plane as a bound surface, but is probably due to thematerials found in the surface. Interestingly, even larger numbersof salmonellae are often observed adhering to surfaces belowthe newly formed outer crust of a gallstone (data not shown).Therefore, in addition to the dependence on bile for efficientSalmonella biofilm formation on gallstones, there appears tobe specificity in attachment to certain gallstone surface structuresor surface components.

Quantification of the bacteria involved in biofilm formationon a gallstone is difficult and inaccurate due to the brittlenature of the gallstone itself, gallstone-to-gallstone variations,and the variable release of bacteria from the stone surfaceby chemical or mechanical means. In order to quantify Salmonelladuring biofilm formation, a system for growing biofilms on glassslides was developed. Biofilm density on the glass slides wasdetermined by the crystal violet staining-destaining methoddescribed in Materials and Methods. Results comparing biofilmformation by wild-type S. enterica serovar Typhimurium in differentmedia (LB broth, LB broth containing 3% bile, TSB, and TSB containing3% bile) are shown in Fig. 3. The results demonstrate that biofilmsare denser and form more quickly when they are grown in TSBthan when they are grown in LB broth. Interestingly, Salmonellais able to form a biofilm on a glass slide in the presence ofLB broth alone, something which does not occur in 14 days ona gallstone, further suggesting that the bacteria interact differentlywith diverse surfaces. In addition, similar to the results observedwith gallstone biofilms, adding bile to either medium (TSB orLB broth) further enhanced biofilm formation on glass slides(Fig. 3).

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FIG. 3. Quantitation of biofilm density. JSG224 was grown in the presence of LB broth alone ( ${blacklozenge}$ ), LB broth containing 3% bile (•), TSB alone ( ${blacktriangleup}$ ), or TSB containing 3% bile ( ${blacksquare}$ ) and assayed quantitatively using the crystal violet staining-destaining method. OD 570, optical density at 570 nm.

Flagella are important for biofilm formation. It has previously been demonstrated that flagella play an importantrole in biofilm formation for organisms such as Escherichiacoli (12, 27), Vibrio cholerae (41), and Pseudomonas aeruginosa(25, 37); in these organisms nonmotile mutants have been shownto be severely defective in the ability to form a biofilm. Flagellaare necessary for moving a bacterium to the surface for initialattachment and across the surface to find other bacteria withwhich to interact. To determine if flagella play a role in Salmonellabiofilm formation on gallstones in our model system, a nonmotilestrain harboring a mutation in fliA was used. fliA encodes thetranscription factor ${sigma}$ ²⁸, which is required for production ofthe late flagellar genes encoding the external filament (8).As with wild-type S. enterica serovar Typhimurium, it was observedthat a fliA mutant did not adhere to gallstones after 4 days.However, while this mutant was found to adhere to gallstonesafter 14 days (albeit in lower numbers), a full biofilm didnot form as it did with the wild-type strain and did not appearto produce the strands and EPS mentioned above (Table 2). Thesedata demonstrate that flagella, although not absolutely required,play a role in the architecture and development of Salmonellagallstone biofilms.

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TABLE 2. Summary of results from SEM visualization of gallstones

The capsule produced by S. enterica serovar Typhi does not play a role in biofilm formation It has been shown that a number of organisms produce an EPSwhen they form a biofilm (11, 12). This polysaccharide is importantfor establishing a matrix within which the organisms are stabilizedand for providing protection against antimicrobial agents andimmune system factors. S. enterica serovar Typhi produces anEPS, Vi antigen (ViAg), which has been implicated in the pathogenesisof typhoid fever (30). This antigen is not produced by S. entericaserovar Typhimurium. Our microscopy studies described abovesuggested that salmonellae form an EPS when they form a biofilm.Because the type of EPS produced by Salmonella spp. within biofilmshas not been defined, an S. enterica serovar Typhi ViAg-deficientstrain (tviB) was examined to determine if the capsule was necessaryfor S. enterica serovar Typhi biofilm formation and responsiblefor the polysaccharide observed by SEM. Gallstones exposed tothis mutant strain for 4 and 14 days showed results identicalto those observed for wild-type S. enterica serovar Typhi (Table2). Few bacteria were observed bound to the gallstone after4 days, and a full biofilm had formed after 14 days. Furthermore,the EPS observed by SEM covering the organisms and the strandsconnecting bacteria to bacteria and bacteria to the gallstonesurface were still present. These results suggest that the ViAgis not the major EPS produced by Salmonella spp. during biofilmdevelopment and does not play a prominent role in biofilm formation.

Salmonellae produce an EPS during biofilm formation. Because, as mentioned above, an EPS produced for biofilm formationhas not been described previously for Salmonella spp. and ViAgof S. enterica serovar Typhi does not appear to play a rolein biofilm formation, it was necessary to confirm the presenceof EPS in the bacterial communities. To confirm that Salmonelladoes produce an extracellular matrix, ruthenium red stain, astain specific for polysaccharides and often used in EPS detection,was used to stain biofilms formed on glass slides. The resultsrevealed ruthenium red staining of regions containing clumpsof cells likely to be in a biofilm or early microcolony formation(Fig. 4). In addition, the gallstones with biofilms were airdried for 4 days before they were placed in the fixing solutionto keep the matrix from condensing during the fixing processfor SEM. This drying procedure preserved the EPS, as shown inFig. 5, in which bacteria appear to be embedded and not simplyon the surface. Also, the web-like strands are not visible,suggesting that they were likely condensed EPS.

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FIG. 4. Salmonella biofilms on glass slides stained with ruthenium red for EPS. (A) Staining biofilms with ruthenium red demonstrated a specificity for groups of bacteria excreting a polysaccharide. Magnification, x100. Bar = 10 µm. (B) Increased magnification of panel A, showing one microcolony stained with ruthenium red. Magnification, x400. Bar = 10 µm.

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FIG. 5. Air drying gallstones before fixation preserves the EPS. A gallstone with an S. enterica serovar Typhimurium biofilm was air dried for 3 days before fixation, and SEM revealed bacteria embedded in a putative EPS matrix without the apparent web-like strands seen in Fig. 2B. Magnification, x7,000.

GalE is necessary for the final stages of biofilm formation. galE, which codes for a uridine diphosphogalactose-4-epimerase,is a structural gene responsible for synthesis of galactose,which is added in both the outer core and the O-antigen. A mutationin this gene produces a lipopolysaccharide (LPS) lacking allsugars beyond the heptose region of the inner core (therefore,a rough or incomplete LPS). A strain containing a Tn10 insertionin galE was examined for its ability to form a biofilm on gallstones.The results demonstrated that the galE mutant could form a biofilmin 14 days but that there were fewer web-like strands and theywere much thinner than the wild-type strands, indicating thatgalE is necessary for the formation of a wild-type biofilm;however, it is not clear if the biofilm deficiency was due tothe lack of LPS O-antigen, EPS production (in which galE alsoplays a role), or both (Table 2).

Fimbriae play a negative role in biofilm formation. It has been demonstrated that in other organisms fimbriae arenecessary for biofilm formation (12, 25, 27, 32). Fimbriae playa role in attachment of the bacteria to other bacteria and tothe surface, and this fimbria-mediated attachment is a signalfor initiation of microcolony formation. Because fimbriae playan important role in biofilm formation in other organisms, variousS. enterica serovar Typhimurium fimbrial mutants were examinedin association with gallstones. Strains with individual mutationsin four fimbrial operons (37), as well as a strain in whichthese four mutations were combined, were examined for gallstonebiofilm formation. These mutations were constructed in S. entericaserovar Typhimurium strain SR11 and were not recreated in ortransduced into the ATCC 14028s background, the parent of allother strains used in this study. Surprisingly, SEM examinationrevealed that all of the fimbrial mutants, including the quadrupleknockout mutant, formed complete biofilms after 4 days (datanot shown). Because an alternate wild-type S. enterica serovarTyphimurium strain was the parent of these fimbrial mutants,SR11 was tested for its ability to form a biofilm on gallstones.It was found that SR11 formed a biofilm indistinguishable fromthose formed by the fimbrial mutants, even after 4 days (bothin the presence and in the absence of bile). Therefore, SR11is much more efficient in the formation of gallstone biofilmsthan ATCC 14028s. Furthermore, in the background of rapid biofilmformation, the fimbriae examined did not appear to play a significantrole in binding to the gallstone surface.

Quorum sensing is important for full biofilm formation. Quorum sensing, which has been shown to be used in many differentbacteria as a mechanism for cell signaling based on cell density,is thought to regulate a variety of processes, such as virulence,motility, conjugation, and biofilm formation (13, 21, 26, 31,33, 34). S. enterica serovar Typhimurium has been shown to havea homologue for luxS, an autoinducer-2 gene (33, 34). The autoinducerhas been implicated in helping the bacteria convert from a pathogenicstate in the host to a free-living state in the environment(33). S. enterica serovar Typhimurium containing a MudJ insertionin luxS was tested for its ability to form biofilms on gallstonesafter 4 and 14 days in the presence of bile. The gallstoneswere viewed by SEM, and no adherent bacteria were found after4 days. The gallstone incubated for 14 days had very few scatteredbacteria with little apparent EPS (Table 2). These results indicatethat quorum sensing is necessary for salmonellae to form a fullbiofilm in the in vitro system employed.

DISCUSSION

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Discussion

Although biofilm formation has been well described for manyorganisms, very little is known about the ability of Salmonellaspp. to form biofilms. While it has been suggested that S. entericaserovar Typhimurium can form biofilms on stainless steel, glass,and polyvinyl chloride (6, 14, 18), the interaction of salmonellaewith organic materials, such as gallstones, has not been examined.In salmonella carriers, formation of a biofilm on gallstonesurfaces would offer the bacteria long-term protection fromantimicrobial agents and high concentrations of bile, as wellas a stable base from which to continually shed planktonic cellsinto the intestine and into the environment.

It has been demonstrated that individuals with gallstones whoare infected with S. enterica serovar Typhi are more likelyto become carriers than individuals without obvious gallbladderabnormalities (15, 20). This suggests that gallstones play arole in carrier state development. Because the gallbladder isconstantly emptying, it would be advantageous for an organismto firmly attach itself to a stable surface, such as a gallstone,to avoid being washed into the environment. Currently, the bestmeans of clearing an S. enterica serovar Typhi carrier statein an individual with gallstones is by surgical removal of thegallbladder or gallstones (20). This suggests that the organismis in a state protected from antibiotics, which further suggeststhe presence of a biofilm.

To determine if biofilm formation can occur on gallstones, humangallstones were grown with Salmonella spp. in the presence ofLB broth containing 3% bile or LB broth alone and examined byboth CSLM and electron microscopy. The results showed that salmonellae(both S. enterica serovar Typhi and S. enterica serovar Typhimurium)formed complete biofilms on gallstones within 14 days in ourmodel system and that bile was required for efficient biofilmformation to occur. Because bile was needed in the medium forsalmonellae to form a biofilm, the following question arose:does bile condition the surface of the gallstone or does itserve as a signal for the bacteria? Experimental results suggestthat conditioning the bacteria or the gallstone with bile isnot sufficient to initiate rapid biofilm formation, indicatingthat an interaction among the gallstone, the bacteria, and thebile is required for biofilm formation in our system. This couldexplain the length of time needed for Salmonella to form a biofilm,as time may be necessary to form the correct environment andfor multiple signals to be sensed and acted upon.

Examination of several different specimens showed that salmonellaehave an affinity for certain surfaces on a gallstone. Salmonelladoes not appear to bind as well to the outermost surface ofa gallstone as to lower layers where the outer crust has notformed yet or has broken off. In addition, two very differentsurfaces can be in one continual plane, yet the bacteria mayadhere to only one of them. This suggests that there is specificityin surface binding, possibly due to a distinct receptor(s) recognizedby the bacterium. To further test the specificity of the organismsfor the gallstone surface, a granite pebble of similar sizewas incubated under growth conditions that were optimal forbiofilm formation on gallstones. The results showed that thebacteria weakly adhere to a pebble under ideal gallstone biofilmconditions, but a typical biofilm is not formed. These resultsfurther suggest that there may be a specific interaction betweenthe gallstone and Salmonella that is necessary to initiate biofilmformation. Because cholesterol and calcium bilirubinate arethe two most abundant components of gallstones, it is possiblethat one of these factors is the ligand bound by the bacterium.

A method for growing biofilms on glass slides was developedin order to have an accurate means of bacterial quantification.Glass slides were incubated in LB broth, LB broth containing3% bile, TSB, and TSB containing 3% bile. The results showedthat all of the media induce biofilm formation (although biofilmsform faster in TSB) and that bile enhances biofilm formationeven more in both LB broth and TSB. These results further confirmthe role of bile in accentuating biofilm formation. In addition,it is of interest that bile is not required for biofilm formationon glass slides but is required for biofilm formation on gallstonesin the system employed. These data again suggest, as mentionedabove, that there is significant interplay among the bacterium,the medium, and the surface that has an impact upon the speedof formation and density of a Salmonella biofilm.

In order to further characterize the ability of salmonellaeto form a biofilm on a gallstone, the roles of several bacterialsurface organelles and products in this process were analyzed.Capsule, flagella, fimbriae, LPS, and quorum sensing were theinitial targets of investigation. Flagella have been shown tobe very important in biofilm formation, especially in the earlystages when microcolonies are being formed. Flagella are neededto move the bacteria to the surface for attachment and thento propel the organisms across the surface in search of otherbacteria (10, 25, 27). Nonmotile E. coli mutants show a severedefect in the ability to form a biofilm (27). To determine ifflagella play a role in Salmonella biofilm formation on gallstones,a mutant that was defective in flagellar production (and thereforenonmotile) was analyzed by SEM. After 14 days of growth, a weakbiofilm was present, but the phenotype was quite different fromthat of the S. enterica serovar Typhimurium wild type. Withthe flagellar mutant, there were many fewer bacteria on thesurface than with the wild type, and they were widely dispersed.Furthermore, EPS was not found to be associated with the nonmotilebacteria bound to the gallstone. These results suggest thatthe Salmonella flagella may play a role in EPS secretion orproduction, as well as in both initial adherence and microcolonyformation (aided by bacterial movement across the surface) ongallstones.

Fimbriae, which have also been shown to be important in biofilmdevelopment, were examined for their role in Salmonella gallstonebiofilms. In E. coli, strains with mutations in type I fimbriaeare severely defective in initial attachment (12, 27). P. aeruginosastrains with a mutation in type IV fimbriae do not form a biofilmas densely packed as that seen with a wild-type strain (25).The type IV fimbriae appear to play a role in full biofilm formationby stabilizing cell-to-cell interactions, and much like flagella,they give the bacteria the ability to move across the surfaceto form multicell aggregates (25). Based upon the genome sequence,S. enterica serovar Typhimurium contains 12 fimbrial operons,most of which have not yet been fully characterized (A. Baumler,personal communication). S. enterica serovar Typhimurium strainswith mutations (either individually or all in one strain) infour of these operons (fim, agf, lpf, and pef) were examinedfor biofilm formation. The fim operon encodes a type I fimbriathat is peritrichous (35, 37). The agf operon encodes a thinaggregative fimbria that is also peritrichous in nature (9,37). The lpf operon encodes long polar fimbriae that are necessaryfor virulence and are needed for colonization of murine Peyer'spatches (3, 5, 37), while the pef operon is on a plasmid andaids in adhesion to the small intestine (4, 37). The S. entericaserovar Typhimurium wild-type strain associated with these mutantsis SR11, while all of the other strains examined in this studywere in an ATCC 14028s background. With each of the fimbrialmutant strains, as well as the SR11 parent, a full biofilm wasformed on gallstones after only 4 days. This time course canbe compared to that of ATCC 14028s, which takes at least 14days to form a complete biofilm. After several days of growthon solid medium, SR11 and the fimbrial mutants appeared to developa rugose-like phenotype. Rugose-colony variants of V. choleraehave been shown to produce an EPS that allows biofilm formation,while smooth-colony variants are deficient for biofilm formation(39, 41). In addition, S. enterica serovar Typhimurium strainDT104 has been shown to exhibit a rugose phenotype on certainmedia, which may accentuate its biofilm-forming ability (1).It is possible that if SR11 can exhibit a rugose phenotype,it may overproduce an EPS that allows rapid biofilm formation.Regardless, because the fimbrial mutants formed biofilms ongallstones indistinguishable from those formed by their SR11parent, none of the fimbriae are likely to be involved in gallstonebiofilms. These data could suggest that different fimbriae areneeded during stages of intestinal and gallbladder infectionand that specific environmental signals, such as bile, may playa significant role in their regulation.

Strains with an incomplete O-antigen or rough LPS have beenshown to be defective in biofilm formation (24). Studies withE. coli that selected for altered or defective biofilm formationrevealed several deep rough mutants (16). A galE mutant, whichis known to be unable to add any sugars to the O-antigen abovethe primary heptose in the core, was examined for its abilityto form a biofilm on gallstones. The results demonstrated thatwhile this mutant is able to adhere and form an early biofilm,it appears to be defective in full biofilm formation. Genevauxet al. suggested that in E. coli, the defective LPS has a pleiotropiceffect on other extracellular structures, such as fimbriae orflagella, and that this is the reason for defective biofilmformation (16). galE has also been shown to be involved in thepathway for producing the sugars needed to make colanic acid,which is an EPS in E. coli that has been implicated in biofilmformation (11, 32). Our results demonstrate that galE playsa role in biofilm formation, but whether it is due to an incompleteLPS or to defective EPS production has not yet been determined.

Many bacteria have developed a type of communication to regulategene expression based on cell density known as quorum sensing(for a review see reference 21). When the autoinducers, andtherefore the bacteria, reach a critical density in an environment,a cascade is initiated in which genes are activated for thecontinued survival of the organism in that environment (13,21, 26, 31, 33, 34). In organisms such as P. aeruginosa, quorumsensing has been shown to be required for both biofilm formationand virulence (13, 21, 26). Autoinducers have been studied inS. enterica serovar Typhimurium and are thought to provide signalsthat aid in modifying the bacteria for survival in the hostversus survival in the environment (33). The best-studied signalingsystem is the acyl-homoserine lactone system (26). Autoinducersfound originally in Vibrio harveyi, a bioluminescent marineorganism, have been divided into two distinct groups, AI-1 andAI-2. AI-1, which is a homoserine lactone, is thought to bea unique signal used only by V. harveyi for intraspecies communication,but AI-2, produced from S-adenosylmethionine, may be used byseveral bacteria for interspecies relations (31, 34). The generesponsible for AI-2 production, luxS, has been shown to behighly conserved in V. harveyi, E. coli, and S. enterica serovarTyphimurium (31, 33, 34). Because these signals have been shownto be important in biofilm formation in other organisms andare present in S. enterica serovar Typhimurium, it was thoughtthat perhaps quorum sensing also plays a role in Salmonellabiofilm formation (13, 21, 26). To test if quorum sensing playsa role in Salmonella biofilm formation, a luxS::MudJ mutantwas examined for its ability to form a biofilm on gallstonesin our model system. The results showed that luxS was necessaryfor biofilm formation, since a biofilm was not formed in 14days by the mutant, in contrast to the wild type. These datasuggest that Salmonella uses quorum sensing (mediated at leastin part by the Salmonella AI-2) to signal full biofilm development.

In addition to analysis of surface organelles and products associatedwith biofilm formation, it is also necessary to further characterizethe extracellular matrix produced by salmonellae in a biofilm.Most bacteria produce an EPS within a biofilm for protectionand stability, but the compositions of the matrices can differgreatly. P. aeruginosa produces an EPS, alginate, that is madeup of glucose, galactose, and pyruvate (29). V. cholerae makesVps, whose primary components are glucose, galactose, N-acetylglucosamine,mannose, and xylose (40). The EPS produced by E. coli, colanicacid, is composed of glucose, galactose, and fucose (11, 32).The EPS produced by Salmonella spp. within a biofilm remainsto be characterized. Through SEM studies of air-dried and non-air-driedgallstones that had been incubated with Salmonella, evidenceof EPS production was obtained. In addition, ruthenium red stainingof biofilms on glass coverslips demonstrated the presence ofpolysaccharide associated with clumps of cells. S. entericaserovar Typhi produces an N-acetylglucosamine uronic acid capsuleknown as ViAg. Because EPS are known to be involved in developmentof the matrix of a biofilm (11, 12), the role of S. entericaserovar Typhi ViAg in gallstone biofilm formation was examined.The results of SEM of gallstones cultured with an S. entericaserovar Typhi tviB strain (for 14 days) showed that the ViAgcapsule does not play a role in biofilm formation and that S.enterica serovar Typhi produces an EPS that is biofilm specific.These results suggest that S. enterica serovar Typhi (and S.enterica serovar Typhimurium as well, as it does not produceViAg) produces another EPS in response to biofilm development.Because E. coli is thought to produce the extracellular polysaccharidecolanic acid within biofilms (11), we are currently examiningwhether a colanic acid-like polymer may be responsible for EPSproduction in Salmonella biofilms.

The results presented here demonstrate that bile is requiredfor salmonellae to form efficient biofilms on gallstones. Severalproteins are both negatively and positively regulated by bile(38). In addition, it has been shown that bile can affect pathogenicproperties of S. enterica serovar Typhimurium, such as invasionof epithelial cells (28). Bile resistance can also be increasedby exposure to sublethal bile concentrations (38). These resultssuggest that salmonellae have the ability to sense bile, andthe presence of bile may lead to induction of bacterial factorsthat promote biofilm formation. These factors could be adhesins(fimbrial or afimbrial) that promote early biofilm formation.In addition to adherence, bile sensing may also result in inductionof bile resistance genes. The ability to adapt to high concentrationsof bile would be important for survival within the gallbladder.Therefore, bile can act as a unique environmental signal, likeclassic environmental signals such as pH, osmolarity, and temperature,to alter bacterial processes. Experiments are currently beingperformed to determine how bile may be sensed in Salmonella,which is clearly necessary for efficient biofilm formation ongallstones in our model system.

We demonstrated that Salmonella can form biofilms on human gallstonesin vitro and that biofilm formation is markedly enhanced inthe presence of bile. It is likely that most, if not all, gallbladdercarriage of Salmonella spp. involves biofilm formation on fullyformed gallstones, newly nucleated pregallstones, or other hepatobiliaryabnormalities. If the process of biofilm formation in the gallbladderis understood, it may be possible to develop therapeutic orpreventative approaches to counteract this chronic infection.

ACKNOWLEDGMENTS

We thank Daniel Guerro for technical assistance with the SEMwork and Vicki Frohlich for assistance with confocal microscopy.Strains used in the study were graciously provided by AndreasBaumler, Michel Popoff, Stanley Falkow, Bonnie Bassler, andKarl Klose.

This work was supported by a grant from the San Antonio AreaFoundation from the Semp Russ Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, MC 7758, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Phone: (210) 567-3973. Fax: (210) 567-3795. E-mail: gunnj{at}uthscsa.edu.

Editor: B. B. Finlay

REFERENCES

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