INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Apr. 1999, p. 1585–1592 Vol. 67, No. 4

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Isolation of Enterococcus faecalis Clinical Isolates That EfficientlyAdhere to Human Bladder Carcinoma T24 Cells and Inhibition

of Adhesion by Fibronectin and Trypsin TreatmentAKIHIKO SHIONO† AND YASUYOSHI IKE*

Department of Microbiology and Laboratory of Bacterial Drug Resistance,Gunma University School of Medicine, Maebashi, Gunma, Japan

Received 15 June 1998/Returned for modification 12 August 1998/Accepted 17 December 1998

The adherence of Enterococcus faecalis strains to human T24 cells was examined by scanning electronmicroscopy. Five highly adhesive strains were identified from 30 strains isolated from the urine of patients withurinary tract infections. No efficiently adhesive strains were found among the 30 strains isolated from the fecesof healthy students. The five isolated strains also adhered efficiently to human bladder epithelial cells. Analysisof restriction endonuclease-digested plasmid DNAs and chromosome DNAs showed that the five strains weredifferent strains isolated from different patients. The adhesiveness of these strains was inhibited by treatmentwith fibronectin or trypsin, implying that a specific protein (adhesin) on the bacterial cell surface mediatesadherence to fibronectin on the host cell surfaces, and the adhesin differs from the reported adhesins.

Enterococci are opportunistic pathogens which cause infec-tions in patients compromised by severe underlying disease,such as urinary tract infections, endocarditis, and wound infec-tions (17, 35, 37, 38, 43, 57). Clinical isolates of enterococci areresistant to many antimicrobial agents in common use (17, 35,38, 44, 53, 57) and thus have a selective advantage in thehospital environment.

Reports describing molecular and genetic studies of entero-cocci pathogenic factors are limited. However, there are anumber of reports that the phenotypes encoded by the Entero-coccus faecalis pheromone-responding plasmids are related topathogenicity. Plasmid pAD1 encodes a b-hemolysin–bacte-riocin (Cyl, cytolysin) mediated by the same genetic determi-nant. A significant number of E. faecalis clinical isolates pro-duce cytolysin (22, 23). More than 50% of the E. faecalisclinical isolates studied carry transferable cytolysin genes (23,24). More than 90% of these cytolysin plasmids are closelyrelated to pAD1 (24, 33). The cytolysin encoded on pAD1 hasbeen shown to enhance the virulence of E. faecalis in animalmodels (3, 25, 29). The transfer functions of the E. faecalispheromone-responding plasmid are induced in the donorstrain by the plasmid-specific peptide sex pheromone which issecreted by the potential recipient cell (5–7, 10). The sex pher-omone induces the synthesis of a surface aggregation sub-stance (adhesin) that facilitates the formation of a matingaggregate (10, 11, 13, 26). The deduced amino acid sequencesof the aggregation substance have extensive homology withsequences of the well-characterized pheromone-respondingplasmids pAD1 (6, 8, 16, 26, 49, 52), pCF10 (4, 12, 21), andpPD1 (11, 15, 16, 55). The aggregation substance of strainscarrying pAD1 has been shown to enhance adherence to renaltubular cell in a cell culture model (28, 30), and it has also beenshown to enhance internalization to cultured intestinal epithe-lial cells (41). Another study has shown that there are two types

of adhesins present on the E. faecalis cell surface (18): (i) aD-mannose-D-glucose-containing adhesin which mediates ad-herence to human urinary tract epithelial cells and humanembryonic kidney cells and (ii) a galactose-containing adhesinwhich mediates adherence to Girardi heart cells and is ex-pressed by strains isolated from patients with endocarditis (18).

In this study, we used scanning electron microscopy forquantitative analysis of adherence to human culture cells ofclinical E. faecalis strains and identified highly efficient adher-ent strains among the clinical isolates.

MATERIALS AND METHODS

Bacteria, media, and reagents. Thirty E. faecalis strains isolated from urinesamples of patients with chronic urinary tract infections were used in this study.Of the 30 strains, 24 were from Gunma University Hospital and 6 were from ahospital in Ota City, Gunma, Japan. Thirty E. faecalis strains isolated from stoolspecimens of 30 healthy students were also used. Laboratory strains used were E.faecalis FA2-2 (Rifr Fusr) (8) and E. faecalis OG1X (Smr) (27). Unless otherwiseindicated, the media used throughout this study were Oxoid Nutrient Broth 2(Oxoid, Basingstoke, Hants, England) supplemented with glucose (0.2%) andTris-hydrochloride (0.1 M, pH 7.7) (N2GT broth). Antibiotic medium 3 (DifcoLaboratories, Detroit, Mich.) was used for testing drug resistance. Antibioticconcentrations (micrograms per milliliter) used in selective plates were as fol-lows: erythromycin, 25; streptomycin, 500; spectinomycin, 500; kanamycin, 500;gentamicin, 200; chloramphenicol, 25; tetracycline, 3; rifampin, 25; and fusidicacid, 25. Hemolysin detection was on Todd-Hewitt agar containing 4% rabbitblood (Toyo Serum Co., Tokyo, Japan).

Bacterial growth condition for adherence. An overnight culture of E. faecalisin N2GT broth was diluted 100-fold with fresh N2GT broth. The diluted bacteriawere grown to an optical density of 200 Klett units (Klett-Summerson colorim-eter; no. 54 filter) at 37°C with slow shaking, and the culture was used foradherence experiments.

Mating procedure. Broth mating was performed as previously described (11,26) with a donor/recipient ratio 1:10. Overnight cultures of 0.05 ml of donor and0.5 ml of recipient were added to 4.5 ml of fresh broth, and the mixtures wereincubated at 37°C with slow shaking for 4 h and then vortexed. Portions of themixed culture were then plated on solid media with appropriate selective anti-biotics, and the plates were incubated at 37°C for 48 h. Filter matings werecarried out as described previously (14) with N2GT broth agar plates containing4% human blood and with an initial ratio of 1 donor per 10 recipients. For thetransfer of hemolysin properties, the mating mixtures were diluted by factors of1021, 1022, and 1024 with fresh N2GT broth. A 0.1-ml sample of each dilutionwas plated on selective Todd-Hewitt agar plates containing 4% human blood andan appropriate drug for counterselection of the donor strains. After overnightincubation of the plates at 37°C, the colonies of recipients producing a hemolyticzone were counted as hemolytic transconjugants.

Isolation and manipulation of plasmid DNA. Plasmid DNA was isolated bythe alkaline lysis method (42). Plasmid DNA was treated with restriction en-

* Corresponding author. Mailing address: Department of Microbi-ology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. Phone: 81-27-220-7990. Fax: 81-27-220-7996. E-mail:yasuike@sb.gunma-u.ac.jp.

† Present address: Department of Urology, Gunma UniversitySchool of Medicine, Gunma, Japan.

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zymes and submitted to agarose gel electrophoresis for analysis of DNA frag-ments, etc. Restriction enzymes were obtained from Nippon Gene (Toyama,Japan) and New England Biolabs, Inc., and were used in accordance with thesuppliers’ specifications. Agarose was obtained from Wako Chemicals, Osaka,Japan.

Pulsed-field gel electrophoresis of the chromosomal DNA. Pulsed-field gelelectrophoresis was performed with a CHEF-DRII system (Bio-Rad, Hercules,Calif.). The embedded chromosome DNAs of E. faecalis strains were preparedand digested according to the manufacturer’s protocols, with some modifications.Cells were embedded in 1% agarose (10 mM Tris-HCl [pH 8.0], 10 mM NaCl, 25mM EDTA) and treated with lysis solution (10 mM Tris-HCl [pH 8.0], 10 mMNaCl, 25 mM EDTA), mutanolysin (150 U/ml; Sigma, St. Louis, Mo.), andlysozyme (8 mg/ml; Sigma) for 2 h. Agarose-embedded chromosome DNA wasdigested overnight with 50 U of SmaI (Nippon Gene) in 300 ml of a 13 dilutionof the recommended reaction buffer.

Clumping assay. Detection of clumping was done as previously described (10).Pheromone corresponded to a culture filtrate of the strains FA2-2. Generally, 1.0ml of culture filtrate from the cells in late log phase was mixed with 1.0 ml offresh N2GT broth and 20 ml of overnight cultured cells to be tested for the abilityto respond. The mixtures were cultured for 4 h at 37°C with shaking and wereexamined for clumping.

Epithelial cells. The T24 cell line, which is derived from a human urinarybladder carcinoma, was kindly provided by the Health Science Research Re-sources Bank (Tokyo, Japan). T24 cells were incubated under 5% CO2 for 24 hat 37°C in Eagle’s minimal essential medium (MEM; GIBCO, Grand Island,N.Y.) supplemented with 10% fetal bovine serum (FBS) and grown withoutantibiotics in a 24-well multidish plate containing a plastic coverslip (SumitomoBakelite; Tokyo, Japan). T24 adhered to a plastic coverslip at 40 to 50% con-fluence.

Specimen of epithelial cells of the human urinary bladder. A specimen ofepithelial cells of the human urinary bladder was obtained by total cystectomyfrom a 68-year-old patient with bladder carcinoma in Gunma University Hospi-tal. The segment, which had no carcinoma, was washed several times with cold(4°C) phosphate-buffered saline (PBS; pH 7.4), and the mucosal side was re-tained. A slice of the mucosa was immediately used for adherence experiments.

Adherence analysis. A previously described method (56) for scanning electro-scopic analysis was modified for direct measurement of adherence of bacteria tohuman epithelial cells. The cells were washed twice with MEM. One milliliter ofMEM without FBS and 40 ml of bacterial culture adjusted to 200 Klett units(Klett-Summerson colorimeter; no. 54 filter) were added to each well, and thenthe plate was incubated for 2 h at 37°C. After incubation, the wells were washedsix times with PBS, and the bacteria adhered to cells on the plastic coverslip werefixed with 2.5% glutaraldehyde in PBS for 3 h (T24 cells) or 72 h (epithelial cellsof the human bladder) at room temperature; postfixing was in 1% osmium

tetroxide for 15 min at room temperature and then for 45 min at 4°C. Thesamples were dehydrated with ethanol, critical point dried, coated with gold-palladium, and examined with scanning electron microscope (S4100; Hitachi,Tokyo, Japan). Adherence of E. faecalis to T24 cells or the plastic coverslips wasobserved; 100 T24 cells or 100 fields of each plastic coverslip were randomlychosen, and the bacteria were counted.

Analysis of inhibitor for adherence. To examine inhibition of the adherence,the samples containing bacteria and the T24 cells were incubated with fibronectin(100 mg/ml) and fibrinogen (500 mg/ml) for 1 h at 37°C. The samples were washedsix times with PBS, fixed, and analyzed by scanning electron microscopy. Fi-bronectin (purified by affinity chromatography of human plasma on gelatin-Sepharose columns) and fibrinogen (purified from human plasma) were pur-chased from Koken (Tokyo, Japan).

Fibronectin treatment of bacteria. Fibronectin was dissolved in PBS andadded to bacterial culture in a final concentration of 100 mg/ml. After 1 h ofincubation at 37°C, the bacteria were washed with PBS and resuspended in PBS.Then 4 ml of the fibronectin-treated bacteria was added to T24 cells in the wellsand incubated for 1 h at 37°C. After incubation, the cells were washed, fixed, andanalyzed for bacterial adherence to the cells.

Trypsin treatment of bacteria. Five microliters of bacterial cultures (200 Klettunits) was heated at 60°C for 10 min, centrifuged (3,000 3 g 15 min), andresuspended in 1 ml of PBS. The suspension was incubated with various con-centrations of trypsin for 30 min at 37°C. The reaction was stopped by theaddition of pancreatic trypsin inhibitor and subsequent washing.

RESULTS

Adherence of E. faecalis strains to T24 cells. The adherenceof E. faecalis strains to T24 cells was examined by scanningelectron microscopy. Each group of 30 E. faecalis strains usedin this study was isolated from the urine samples of patientswith urinary tract infections or the feces of healthy students.For most of the E. faecalis strains derived from urine samples,the number of adherent bacterial cells was less than 40 per T24cell (Fig. 1A). Five strains (AS11, AS12, AS13, AS14, andAS15) adhered more efficiently than the other strains to T24cells. Typical results are shown in Fig. 2. The number of ad-herent cells observed for these strains was more than 230 per462 mm2 (the average area of a T24 cell). The number ofbacterial cells adhering to T24 cells in the efficiently adhesive

FIG. 1. Adherence of E. faecalis strains to T24 cells (A) and to plastic coverslips (B). Each circle represents an E. faecalis strain. Laboratory strains used were FA2-2,OG1X, FA2-2(pAD1), and OG1X(pAD1). Total numbers of adherent bacterial cells per 462 mm2 (the average area of a T24 cell) are shown.

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strains was significantly higher [F(2.57) 5 24.85, P , 0.0001(analysis of variance); P , 0.001 (Fisher’s PLSD)] than thoseof the inefficiently adhesive strains.

For strains isolated from the feces of healthy students, thenumber of adherent cells was less than 20 per 462 mm2 (Fig.1A). Figure 3 shows typical results for inefficiently adhesivestrains isolated from urine and fecal samples.

Adherence of E. faecalis strains to human bladder epithelialcells. Adherence of the efficiently adhesive strains to epithelial

cells of the human urinary bladder was examined as describedin Materials and Methods. The efficiently adhesive strains alsoefficiently adhered to the epithelial cells. Typical results for theadherence of strains AS11 and AS12 are shown in Fig. 4. Onthe other hand, the inefficiently adhesive strains AS21 andAS23, which were derived from urine and feces, respectively,did not adhere to the epithelial cells (data not shown).

Adherence of E. faecalis strains to plastic coverslips. Theadherence of E. faecalis strains to the plastic coverslips used forculture of the T24 cells was also examined by scanning electronmicroscopy. With a few exceptions, fewer than 20 bacterialcells of each strain per 462 mm2 adhered to the plastic cover-slips (Fig. 1B). As indicated above, strains AS11, AS12, AS13,AS14, and AS15 efficiently adhered to T24 cells. On the otherhand, the number of these cells adhering to the plastic cover-slip was low, ranging from 15 to 25 per 462 mm2. For theefficiently adhesive strains, the number of bacterial cells ad-hering to T24 cells was significantly higher (t 5 27.64, 4 df, P ,0.0001) than the number adhering to the plastic coverslips.These results imply that these strains have a specific mecha-nism for adherence to the host cell surface.

Clinical surveillance of patients. We monitored the clinicalcourse of 9 of the 30 patients. Six of the nine patients had beeninfected with inefficiently adhesive strains, and E. faecalis wasnot detected in their urine beyond the first month of follow-up.Each of the other three patients had been infected with one ofthe efficiently adhesive strains (AS13, AS14, or AS15), and E.faecalis was detected in their urine during the follow-up sur-veillance for a period of 12, 7, or 3 months, respectively. Thechromosomal DNA patterns of the E. faecalis isolates obtainedfrom each of the three patients at different times during fol-low-up were examined by pulsed-field gel electrophoresis. Thepatterns obtained by pulsed-field gel electrophoresis of SmaI-digested chromosomal DNAs from the E. faecalis isolates ob-tained from the same patient were identical (data not shown),which implied that the efficiently adhesive strains producedinfection for a longer period of time than the inefficientlyadhesive strains.

Conjugative plasmids of the efficiently adhesive strains.Three (AS11, AS12, and AS13) of the efficiently adhesivestrains contained plasmids (data not shown). AS11 was a pher-omone-responding strain which aggregated by exposure to aculture filtrate of E. faecalis FA2-2 (data not shown). AS13 wasa constitutive-clumping strain. The transferabilities of the plas-mids to FA2-2 or OG1X were examined by mating experi-ments.

The plasmid isolated from AS11 had an EcoRI profile al-most identical to that of the conjugative cytolysin plasmidpAD1 (60 kb) (data not shown) (8). The transferability of theb-hemolytic trait (cyl) of AS11 was examined as described inMaterials and Methods. The b-hemolytic trait transferred torecipient strains at a frequency of approximately 1023 perdonor cell. The tetracycline resistance trait did not transfer bybroth mating. The plasmid of the b-hemolytic transconjuganthad an EcoRI profile similar to that of the plasmid of the AS11donor strain. The transconjugant was aggregated by exposureto FA2-2 culture filtrate (pheromone).

AS12 transconjugants were obtained by filter mating andselected on the basis of tetracycline resistance. The EcoRIrestriction fragments of the transconjugant plasmid DNAswere examined by agarose gel electrophoresis. The restrictionendonuclease digestion patterns of the plasmids showed twodifferent patterns. The molecular size of one plasmid was 45.8kb, and that of the other plasmid was 61 kb. The 45.8-kbplasmid consists of eight EcoRI fragments with molecular sizesof 12.0, 10.4, 8.9, 5.2, 3.8, 3.4, 1.4, and 0.7 kb. The 61-kb

FIG. 2. Adherence of efficiently adhesive strains AS11 (A), AS12 (B), andAS15 (C) to T24 cells.

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plasmid also consists of eight EcoRI fragments, in this casewith molecular sizes of 24.1, 12.0, 10.4, 5.2, 3.8, 3.4, 1.4, and 0.7kb. The difference in plasmid sizes was due to the 8.9-kbfragment of the 45.8-kb plasmid and the 24.1-kb fragment ofthe 61-kb plasmid. A second mating experiment with theFA2-2 donor strain containing either the 45.8-kb or the 61-kbplasmid and the recipient strain OG1X was performed. The

EcoRI restriction fragments of the 10 tetracycline resistancetransconjugants obtained in each mating experiment were ex-amined by agarose gel electrophoresis. Seven of the tentransconjugants derived from the mating experiment with thedonor strain containing the 45.8-kb plasmid contained the45.8-kb plasmid. Three of the ten transconjugants containedthe 61-kb plasmid in the corresponding experiment using the

FIG. 3. Adherence of inefficiently adhesive strains AS21 (A), AS22 (B), AS23 (C), and AS24 (D). AS21 and AS22 were isolated from urine samples; AS23 and AS24were isolated from feces.

FIG. 4. Adherence of efficiently adhesive strains AS12 (A) and AS11 (B) to epithelial cells of the human urinary bladder.

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61-kb plasmid. All transconjugants obtained in the mating ex-periment with the donor strain containing the 61-kb plasmidcontained the 61-kb plasmid. These results suggested thatAS12 harbored a 45.8-kb conjugative plasmid and a tetracy-cline resistance conjugative transposon of approximately 15 kb.

The 61-kb plasmid resulted from the insertion of the 15-kbconjugative transposon into the 45.8-kb conjugative plasmid.The E. faecalis strain containing the 45.8- or 61-kb plasmid wasnot aggregated by exposure to a FA2-2 culture filtrate (pher-omone).

The AS13 transconjugants were obtained by broth matingand were selected on the basis of tetracycline resistance. Thetetracycline resistance transconjugants of AS13 were isolatedat a frequency of 1022 to 1023 per donor cell. The transcon-jugants also showed constitutive clumping and contained the61.4-kb plasmid.

The molecular size of the plasmid contained in eachtransconjugant is shown in Table 1. Each of the FA2-2 orOG1X transconjugants was examined for adherence to T24human culture cells and found not to efficiently adhere to thecells (data not shown).

Restriction endonuclease digestion patterns of the E. faeca-lis chromosomal DNA. Pulsed-field electrophoresis was used tocompare the efficiently adhesive strains. The restriction endo-nuclease digestion patterns of the five E. faecalis chromosomalDNAs showed five different patterns (Fig. 5). Two strains,AS11 and AS13, had almost identical restriction endonucleasedigestion patterns; however, the sizes of the two largest bandsin these plasmids differed slightly. AS11 and AS13 were iso-lated from different patients in geographically distant hospi-tals. AS11 was a tetracycline-resistant, b-hemolytic, phero-mone-responsive strain; AS13 was a tetracycline-resistant,constitutive-clumping strain. These results indicate that AS11and AS13 are different strains.

Inhibition of adherence by fibronectin. To examine if fi-bronectin or fibrinogen inhibits adherence of the efficientlyadhesive strains, we added each compound to a mixture of E.faecalis AS11 or AS12 and T24 cells, and then examined ad-herence by scanning electron microscopy. Fibronectin, but notfibrinogen, inhibited the adherence of E. faecalis AS11 or AS12to T24 cells (data not shown). The adherence of E. faecalisOG1X was not affected by these compounds (data not shown).The effect of fibronectin, an epithelial cell compound, on ad-herence was examined by pretreatment of E. faecalis strainswith fibronectin. When E. faecalis AS11 or AS12 was preincu-bated with fibronectin, adherence to T24 cells was inhibited(Fig. 6 and 7).

Inhibition of adherence by protenase. To examine whetherprotenase affects adherence, E. faecalis AS11 or AS12 waspreincubated with trypsin and then examined for adherence.As shown in Fig. 8, the number of trypsin-treated E. faecalisstrains adhering to the T24 cells decreased in proportion to thetrypsin concentration used in the preincubations.

DISCUSSION

E. faecalis strains are frequently isolated from urine and aremajor causative agents of chronic urinary tract infections. Useof scanning electron microscopy for quantitative analysis of theadherence of E. faecalis clinical strains made it possible toidentify from among strains isolated from urinary tract infec-tions those that were highly adhesive to human culture cells.The highly adhesive strains were shown to be different strainsisolated from different patients. On the other hand, efficientlyadhesive strains were not isolated from the 30 strains derivedfrom feces of healthy students. The isolation frequency ofhighly adherent strains from the urine of patients with urinarytract infections was significantly greater (P 5 0.0261, Fisher’sexact method) than that found for strains derived from thefeces of healthy students.

The highly adhesive strains adhered efficiently to the cul-tured cells and the human bladder cells but not to plasticcoverslips. The other strains examined adhered equally to cul-ture cells and plastic coverslips. These observations impliedthat the highly adhesive strains have specific substances on thebacterial cell surface which produce adherence to the hostepithelial cell surface. The gram-positive bacteria Streptococ-cus pyogenes and Staphylococcus aureus bind to extracellular

FIG. 5. Pulsed-field gel electrophoresis of SmaI-digested chromosomalDNAs isolated from efficiently adhesive strains. Lane 1, AS11; lane 2, AS12; lane3, AS13; lane 4, AS14; lane 5, AS15; lane 6, OG1X; lane 7, FA2-2; lane 8,bacteriophage lambda DNA ladder.

TABLE 1. Efficiently adhesive strains

E. faecalis strain Phenotype Plasmid content Conjugative plasmid identified (size), phenotype conferred by plasmid

AS11 Cyl Tetr pheromone response 1 pAS11 (60 kb), Cyl pheromone responseAS12 Cyl Tetr 1 pAS12 (45.8 kb)AS13 Tetr constitutive clumping 1 pAS13 (61.4 kb), Tetr constitutive clumpingAS14 2AS15 Tetr 2

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host proteins that are present on the surface of the target hostcells. The host proteins include components of the extracellu-lar matrix, such as fibronectin (1, 20, 31, 32, 39, 45), fibrinogen(9, 36, 54), laminin (34, 48), collagen (46, 47), and vitronectin(2, 50). The binding of bacteria to these host proteins is me-diated by specific proteins on the bacterial cell surface (9, 19,20, 40, 51, 54).

We investigated whether a protein on the bacterial cell sur-face of the efficiently adhesive strains mediates adherence tothe host protein. The adherence of the bacterial cells wasinhibited by pretreatment of the bacterial cells with proteinase(trypsin) and fibronectin. The results imply that a specific pro-tein (adhesin) on the bacterial cell surface mediates adherenceto fibronectin on the host cell surfaces. These results show thatthe adhesin differs from the reported E. faecalis adhesins (18,28, 30), i.e., the D-mannose-D-glucose-containing adhesin (18),the galactose-containing adhesin (18), and the aggregationsubstance encoded on the pheromone-responsive plasmid (30).

The D-mannose-D-glucose-containing adhesin was expressedin all E. faecalis strains examined which are involved in urinarytract infections and endocarditis (18). Thus, adhesin could bea widespread or general adhesin. In our study, the inefficientlyadhesive strains isolated from urine and feces adhered to bothculture cells and plastic coverslips to the same degree. It ap-pears that the adherence of these strains is mediated by ageneral or widespread adhesin, such as the D-mannose-D-glu-cose-containing adhesin.

The role of the adhesin in the pathogenicity of the efficiently

FIG. 6. Adherence of fibronectin-treated (A) and untreated (B) E. faecalisstrains to T24 cells. The efficiently adhesive strains AS11 and AS12 were treatedwith fibronectin for 1 h at 37°C and then examined for the adherence to T24 cells.

FIG. 7. Adherence of untreated strains AS11 (A) and AS12 (B) and of fibronectin-treated strains AS11 (C) and AS12 (D) to T24 cells.

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adhesive strains is not clear. The study of E. faecalis strainsisolated from patients during clinical surveillance showed thatpatients who had been infected with efficiently adhesive strainshad been infected for a longer period than patients infectedwith the inefficiently adhesive strains, which implies a correla-tion between the difficulties observed in the treatment of casesand infection with efficiently adhesive strains.

Two of the highly adhesive strains harbored pheromone-responsive or constitutive aggregation plasmids. Neither ofthese plasmids, when transferred into the laboratory strain E.faecalis FA2-2 or OG1X, conferred the efficiently adhesivephenotype. These results suggest that the aggregation sub-stance encoded on these plasmids plays little role in efficientadherence, although it is possible that the original host deter-minant was required to act together with a plasmid determi-nant for expression of the adherence characteristics of thestrains harboring the conjugative plasmids.

ACKNOWLEDGMENTS

This work was supported by grants from the Study of Drug ResistantBacteria funded by the Ministry of Health and Welfare, Japan, in 1996,1997, and 1998 and by the Japanese Ministry of Education, Scienceand Culture.

We thank H. Yamanaka and Y. Fukabori (Department of Urology,Gunma University School of Medicine) for helpful advice and forproviding T24 cells, and we thank E. Kamei for helpful advice on themanuscript.

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FIG. 8. Adherence of trypsin-treated E. faecalis strains to T24 cell. The efficiently adhesive strains AS11 (E) and AS12 (F) were treated with various concentrationsof trypsin for 30 min at 37°C and then examined for adherence to T24 cells.

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