Vol. 56, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1926-19310099-2240/90/061926-06$02.00/0Copyright © 1990, American Society for Microbiology

Particle Agglutination Assays To Identify Fibronectin and CollagenCell Surface Receptors and Lectins in Aeromonas

and Vibrio SpeciesF. ASCENCIO,"2 P. ALELJUNG,l AND T. WADSTROM1*

Department of Medical Microbiology, University ofLund, Solvegatan 23, S-223 62 Lund, Sweden,' and Department ofExperimental Biology, Centro de Investigaciones Biologicas de Baja California Sur, La Paz, Baja California Sur, Mexico2

Received 30 November 1989/Accepted 25 March 1990

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serumproteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, andtransferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, humaninfections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as wellas Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV andfibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastricmucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reactionwas studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions insolutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-richglycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connectivetissue proteins was found to depend on culture methods.

Aeromonas salmonicida is an important fish pathogen,while Aeromonas hydrophila and a number of Vibrio speciesare pathogens for both fish and other warm-blooded animals,including humans (8, 9, 22, 28). Most of these microorgan-isms produce a variety of hemolysins or cytolytic toxins andsome (such as Vibrio cholerae) also produce cytotoxicenterotoxins such as choleragen (18, 24, 26).

Studies by Atkinson and Trust (1) have shown that A.hydrophila produces a variety of surface hemagglutinins.Faris et al. (4) and others (11, 16, 27) showed that V.cholerae and other vibrios can produce several hemaggluti-nins. Moreover, Wiersma et al. (29) showed that V. choleraebinds specifically to one serum and connective tissue pro-tein, i.e., fibronectin.We have recently developed a particle agglutination assay

(PAA) for rapid screening of bacterial cell surface receptorswith biospecific affinity to fibronectin (Fn), fibrinogen (Fg),and collagen (Cn) (21). We now report on the evaluation ofsuch Fn, Fg, and types I, II, and IV Cn (Cn-I, Cn-II, Cn-IV)PAAs, as well as similar tests with various glycoprotein PAAreagents for rapid screening of cell surface receptors instrains of various Aeromonas and Vibrio species isolatedfrom diseased fish, human stools, and the environment.

MATERIALS AND METHODSChemicals. Porcine plasma Fn was a kind gift from Bioln-

vent International AB, Lund, Sweden. Highly purified hu-man Fg (batch 61857) was kindly supplied by Kabi, Stock-holm, Sweden. Orosomucoid (a-1-glycoprotein concentratefrom human plasma, batch FVII 12) was a kind gift from theScottish National Blood Transfusion Association ProteinFractionation Center, Edinburgh. Dextran (Dx)-palmitate,DEAE-Dx, and polyethylene glycol (PEG)-palmitate werekind gifts from Gote Johansson, Department of Biochem-istry, University of Lund. Cn-IV from human placenta(C-7521; lot 52F-3848), gelatin (G-2500; lot 64C 0077), trans-

* Corresponding author.

ferrin from rabbit plasma (T-6136), ovalbumin grade V(A-5503), bovine fraction VI glycoproteins (G-3259), crudemucin from porcine stomach (type II [M-2378]), asialomucinfrom bovine submaxillary gland (A-0789), fetuin, fraction IV(F-3004), and asialofetuin from fetal calf serum (type I[A-4781]) were purchased from Sigma Chemical Co., St.Louis, Mo. All other chemicals used were purchased fromdifferent commercial sources and were of analytical grade.Latex suspensions (control 764522) and the bacteriologicalmedia used were purchased from Difco Laboratories, De-troit, Mich.

Bacterial strains and culture conditions. Strains used in thisstudy were isolated from diseased fish, human infections,and the environment (a complete list of the bacteria strainsused can be obtained from the corresponding author).Strains were grown on blood agar for 24 h for all bindingassays. Bacterial colonies were suspended and washed oncein 0.02 M potassium phosphate buffer (pH 6.8). Bacterialcells were resuspended in the same buffer to approximately1010 cells per ml and immediately used in various bindingassays. The same cell suspensions were used for the saltaggregation test (SAT).

Preparation of standard latex reagents. Standard latexreagents were prepared and used as previously described(21). Briefly, 1 ml of latex particle suspensions (bead diam-eter of 0.8 ,um) was mixed with 3.0 ml of 0.17 M glycine-NaOH buffer (pH 8.2) and centrifuged at 4,500 x g for 5 min,and the cell pellets were resuspended in 3.0 ml of the samebuffer. Highly purified protein (100 ,ug) was added, and themixtures were kept at 30°C for 12 h on a horizontal shaker at50 rpm. The mixtures were centrifuged (9,200 x g for 5 minat 20°C), and the supernatants were discarded. Pellets weresuspended in 2.0 ml of the glycine buffer containing 0.01%ovalbumin and 0.01% Merthiolate and kept at 4°C for 12 h.A gelatin reagent (gelatin PAA) was prepared as described

above with a slight modification. Gelatin (200 ,ug) was addedto 0.2 ml of glycine buffer, dissolved by warming at 45°C for

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10 min in a water bath, and then added to latex suspensions.The reagents were thoroughly suspended before use.PAA. PAA was performed as previously described (21).

Briefly, latex reagents (20 ,ul) were placed on a glass slide,and equal volumes of bacterial cell suspensions were added.The two drops were gently mixed, and the agglutinationreaction was read after 2 min. The reactions were scored(PAA value) from strongly positive (3) to weakly positive (1)or negative (0) as previously described (21).

Strains were tested for autoaggregation by mixing 1 dropof bacterial cell suspension with 1 drop of potassium phos-phate buffer.

Bacterial cell suspensions of Staphylococcus aureusCowan 1 and Newman were used as positive controls andstrain Wood 46 was used as a negative control for the Fn,Cn, Fg, and gelatin PAAs (21).

Particle agglutination inhibition assay. PAA reactivity wasblocked with soluble gelatin and bovine serum albumin(BSA) (at final concentrations of 0.12, 0.25, 0.5, and 1.0mg/ml) or with glycoproteins and carbohydrates (0.1% fe-tuin, bovine serum glycoprotein, bovine and porcine mucin,orosomucoid, or 0.1 M N-acetyl-D-galactosamine). Briefly,100 p,l of inhibitor was preincubated with an equal volume ofa bacterial cell suspension as in the standard PAA for 30 minat 20°C and then mixed with the PAA reagents. Test reac-tions were scored as in the standard PAA test (21).SAT. SAT was performed as previously described (18).

Bacterial cells were suspended in 0.001 M sodium phosphatebuffer (pH 7.0), washed once in this buffer, and diluted to1010 bacteria per ml. From a series of ammonium sulfatesolutions of different molarities (0.02 to 4.0 M, pH 6.8), fourconcentrations, 0.02, 0.2, 2.0, and 4.0 M, were chosen asrepresenting breakpoints for groups with different hydropho-bicity. Bacterial suspensions (20 ,ul) were mixed with equalvolumes of ammonium sulfate solutions of various molarityon a glass slide. The highest dilution of ammonium sulfate(final concentration) which gave visible aggregation in 2 minwas scored as a numerical value for bacterial cell surfacehydrophobicity (the SAT value).

Partitioning in an aqueous two-phase system. An Aqueouspolymer two-phase system containing PEG 6000 (Kebo)(7.13%, wt/wt) and Dx (molecular weight, 48,000; Sigma)(8.75%, wt/wt) in 0.015 M NaCl (pH 6.8) was prepared as aphase system (10). To determine particle negative charge,negatively charged Dx sulfate (Pharmacia, Uppsala, Swe-den) at a concentration of 0.40% (wt/wt) was included in thephase system, replacing an equivalent amount of Dx. Simi-larly, positively charged DEAE-Dx at a concentration of0.40% was included, replacing an equal amount of Dx in thephase system.Hydrophobic affinity partitioning was performed by in-

cluding monosubstituted PEG-palmitate and Dx-palmitate,replacing an equal amount ofPEG (for the PEG-palmitate) orDx (for the Dx-palmitate) in the phase system. Partitioningwas performed by adding 100 ,ul of coated latex particles(absorbance of 1.0 at 540 nm) to 0.9 ml of the phase systems(previously homogenized by simple stirring), mixing bygently shaking, and allowing phase separation at 20°C for 1h. The concentration of latex particles in the PEG-rich topphase and the Dx-rich bottom phase was then estimatedturbidimetrically at 540 nm, and beads recovered in the topphase were expressed as a percentage of the original con-centration of added latex beads. Differences in the hydro-phobic and charge properties of the various coated latexbeads are expressed as Alog G, which is defined by theequation:

TABLE 1. Distribution of immobilized connective tissue andglycoprotein binding to Vibrio and Aeromonas strains by the PAA

% Strains showingpositive reactivity

Latex beads coated with: (no. of strains tested)

Aeromonas sp. Vibrio sp.

Connective tissue proteinCn-I 83 (147) 58 (33)Cn-II NDa 88 (33)Cn-IV 69 (147) 90 (29)Fn 78 (49) 44 (34)

Fg 58 (147) 93 (29)Gelatin 55 (147) 70 (33)

GlycoproteinBovine submaxillary mucin 53 (49) 58 (36)Mucin (crude porcine stomach type II) 69 (49) 78 (32)Hog gastric mucin 59 (49) 41 (32)Asialomucin (bovine submaxillary 40 (20) 19 (27)

gland)Orosomucoid (human plasma) 63 (49) 44 (35)Fetuin (calf serum, fraction V) 30 (49) 20 (25)Asialofetuin (calf serum, type I) 63 (49) 74 (35)Glycoprotein (bovine, fraction VI) 37 (49) 39 (31)Transferrin (rabbit) 40 (20) 4 (28)

Ovalbumin (albumin, egg) 53 (147) 6 (36)BSA 55 (147) 7 (27)

Without protein 55 (147) 6 (36)a ND, Not determined.

G value of Dx-sulfate system (for Alog G charge) orG value of PEG-palmitate system (for Alog G hydrophobicity)

Alog G = logG value of the PEG-Dx system

percent cells in top phasewhere G =

percent cells in rest of system

12'I-labeled connective tissue protein binding assay. Sam-ples of 50 ,ug of Fn, Cn-I, Cn-IV, and Fg were labeled with0.2 mCi of 1251 by the method described in reference 19 withlodo-beads.

Binding of 1251-connective tissue proteins to Aeromonasand Vibrio strains was quantitated as previously described(5). S. aureus Cowan 1 and Newman served as positivecontrols, and strain Wood 46 served as the negative control(21).

RESULTS

A selected number of Aeromonas and Vibrio strains weregrown in and on various broth and agar media, respectively.Cell suspensions were then tested with a series of PAAs: FnPAA, Fg PAA, Cn-I PAA, and Cn-IV PAA (Table 1).Identical experiments were conducted for latex particlescoated with a series of glycoproteins (Table 1). BSA (0.1%)was added to the standard PAA buffer to prevent nonspecificbinding.

Cells of Vibrio strains generally bound to latex beadscoated with Cn-II, Cn-IV, and Fg and, to a lesser degree, tobeads coated with Cn-I, gelatin, and Fn (Table 1). Cells ofthese Vibrio strains did not agglutinate ovalbumin- or serum

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TABLE 2. Inhibition of binding of connective tissue protein-coated latex beads to Vibrio and Aeromonas strains by gelatin or BSA

Reaction with the following PAA reagenta:Strain incubated with: Without SAT value

Cn-I Cn-IV Fn Fg Gel Ov BSA protein

Vibrio cholerae 635-85BSA (mg/ml)

10.0 0 0 0 0 0 0 0 0 2.05.0 0 1 1 1 0 0 0 0 2.02.5 1 1 1 2 1 1 1 1 2.01.25 1 1 1 2 1 1 1 1 2.0

Gelatin (mg/ml)10.0 0 0 0 1 0 0 0 0 1.05.0 0 0 0 1 1 0 0 0 1.02.5 0 1 0 1 1 1 0 0 1.01.25 0 1 0 1 1 1 0 0 1.0

Control (without inhibitor) 2 1 2 3 3 2 2 1 2.0

Aeromonas sobria AS-76BSA (mg/ml)

10.0 3 0 1 3 1 2 2 2 1.05.0 3 0 2 3 1 3 3 3 1.02.5 3 0 3 3 2 3 3 3 1.01.25 3 0 3 3 3 3 3 3 1.0

Gelatin (mg/ml)10.0 0 0 0 0 0 0 0 0 1.05.0 1 1 0 1 1 0 0 0 1.02.5 1 1 0 1 1 1 0 1 1.01.25 1 1 1 1 2 1 1 2 1.0

Control (without protein) 3 1 3 3 3 3 3 3 1.0

Aeromonas caviae E46985BSA (mg/ml)

10.0 0 0 0 0 0 0 0 0 2.05.0 0 0 0 0 0 0 0 0 2.02.5 0 0 0 0 0 0 0 0 2.01.25 0 0 0 0 0 0 0 0 2.0

Gelatin (mg/ml)10.0 0 0 0 0 0 0 0 0 1.05.0 0 0 1 1 1 0 0 1 1.02.5 1 0 1 2 1 1 0 1 1.01.25 1 0 1 2 1 1 0 1 1.0

Control (without protein) 2 0 1 2 1 1 0 1 2.0

a See text for definition of reaction values. Gel, Gelatin; Ov, ovalbumin.

albumin (BSA)-coated latex beads or uncoated latex beads.In contrast, Aeromonas strains reacted equally well with allthe proteins tested, with the highest affinity observed forCn-I-coated latex beads (Table 1).

Cells of various Vibrio and Aeromonas strains commonlyagglutinated glycoprotein-coated latex beads (Table 1), withthe exception of Vibrio strains and ovalbumin- or transferrin-coated latex beads.

Strains showing different reactions in a number of PAAswere investigated for surface hydrophobicity by the SATand by phase partitioning. There was no correlation betweenbacterial cell surface hydrophobicity and/or charge and thePAA reactivity (unpublished data).

Autoaggregating strains were grown in broth and on agarmedia to define conditions which inhibit expression of cellsurface hydrophobicity without affecting the reactions inPAAs. However, no simple procedure to suppress nonspe-cific hydrophobic cell properties could be defined.

Agglutination inhibition of latex beads coated with Cn-I,Cn-IV, Fg, gelatin, and Fn could be produced by preincu-bating Vibrio cells with gelatin (i.e., denatured Cn) beforeadding the PAA reagents and, to a lesser degree, by prein-

cubating the bacterial cells with BSA (concentration up to 5mg/ml) (Table 2). Similarly, agglutination inhibition of latexbeads coated with the various glycoproteins could be pro-duced by preincubating bacterial cells with bovine serumglycoproteins or fetuin and, to a lesser degree, with oroso-mucoid, asialofetuin, and N-acetyl-D-galactosamine butcould not be produced with bovine submaxillary mucin orporcine stomach mucin (Table 3).

Differences in 251I-labeled Cn-I and Fg binding to V.cholerae 635-85 grown under various conditions were ob-served (Table 4). Similar results were obtained with Aero-monas strains of different species (data not shown). Thehighest 125I-labeled Cn-I binding to V. cholerae 635-85 wasobtained with cells grown on blood agar incubated at 25°C.Bacteria grown in peptone water incubated at 25°C boundmore 125I-labeled Fg.PAA reagents were tested for surface hydrophobicity and

charge properties by partitioning in aqueous two-phasesystems of Dx and PEG. In general, uncoated and coatedlatex beads possessed both hydrophobic and charged surfaceproperties (Table 5).

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TABLE 3. Inhibition of binding of glycoprotein-coated latexbeads to cells of V. cholerae 635-85 by soluble glycoproteinsa

Reaction with the following PAA reagentb:Strain 635-85

incubated with: Ft AsFt BSM Mucin HGM OM p rWithout(Ps) (b) protein

Control (without 0 2 2 2 2 2 1 2 1inhibitor)

Fetuin 0 1 0 1 0 0 0 0 0Asialomucin 0 1 1 1 1 1 1 1 1BSM 1 2 2 2 2 2 2 2 1Mucin (ps) 1 2 2 2 2 2 2 2 2HGM 0 1 0 1 1 0 0 0 0OM 1 2 1 1 2 1 1 2 2Gp (b) 0 0 0 0 0 0 0 0 0Ovalbumin 1 2 2 2 2 2 1 1 1N-Acetyl-D-ga- 0 1 1 1 1 0 0 0 0

lactosamineTween 20 (0.5%) 0 0 1 0 0 0 0 0 0

a Abbreviations: AsFt, asialofetuin (calf serum, type I); BSM, mucin(bovine submaxillary gland); Mucin (ps), crude, porcine stomach type IImucin; HGM, hog gastric mucin; OM, orosomucoid (human plasma); Gp (b),glycoproteins (bovine serum, fraction VI). The concentration of inhibitor was0.1% for glycoproteins and 0.1 M for carbohydrate.

b See text for definition of reaction values.

DISCUSSION

Immobilizing various immunoglobulins on latex beads is astandard procedure in particle immunoassays for rapid de-tection of bacterial capsular and other surface antigens invarious body fluids during infections (20). More recently, anumber of PAAs have been developed, such as Fg-immuno-globulin latex tests, for rapid diagnosis of staphylococci withcell surface Fg receptors and protein A (i.e., S. aureus) andof other closely related species (3, 21).

Accurate, reproducible, and rapid results obtained byusing the PAA to screen staphylococci for Fg, Fn, and Cn-Iand Cn-II cell surface receptors (21) encouraged us toevaluate the same assays for testing Vibrio and Aeromonasstrains for similar cell surface receptors. Since Aeromonasand Vibrio species commonly produce a number of cellsurface lectins, we expanded this list of PAA reagents byalso coating latex beads with various glycoproteins, i.e.,bovine and porcine mucins, orosomucoid, fetuin, and bovineserum glycoproteins (Table 1).

TABLE 4. Effect of culture medium and growth temperature on1251I-labeled Cn-I and Fg binding to V. cholerae 635-85

% 125I-protein binding at the followinggrowth temp:

Growth medium Cn-I Fg

250C 32°C 370C 250C 320C 370C

CFA 18.0 4.0 8.0 10.0 9.0 0.0MacConkey agar 4.0 10.0 8.0 0.0 0.0 0.0Tryptic soy agar 30.0 6.0 19.0 12.0 15.0 8.0Blood agar 32.0 5.0 20.0 9.0 10.0 11.02216 agar 13.0 19.0 6.0 0.0 12.0 11.0TCBS agar 5.0 5.0 9.0 0.0 9.0 9.0Peptone water 30.0 22.0 8.0 18.0 9.0 0.0Brain heart infu- 13.0 22.0 19.0 9.0 12.0 10.0

sion broth2216 broth 16.0 12.0 10.0 9.0 8.0 7.0Tryptic soy broth 16.0 22.0 NDa 5.0 9.0 ND

a ND, Not determined.

TABLE 5. Partitioning behavior of various PAA reagents inaqueous two-phase system of PEG-Dx

ALog GLatex beads coated with: Hydro-

Charge phobicity

GlycoproteinBovine submaxillary mucin 0.83 0.27Mucin (crude, porcine stomach, type II) 0.14 0.00Hog gastric mucin 0.19 0.25Asialomucin (bovine submaxillary gland) 0.24 0.70Orosomucoid (human plasma) 1.05 2.04Fetuin (calf serum) 0.75 0.68Asialofetuin (calf serum, type I) 0.74 2.01Glycoprotein (bovine fraction VI) 0.24 1.16Transferrin (rabbit) 0.63 1.64BSA 0.84 0.81Ovalbumin (egg albumin) 0.65 0.62

Connective tissue proteinCn-I 0.17 -0.08Cn-IV 0.20 0.37

Gelatin 0.52 1.09Fg (human) 0.52 0.62

Without protein 0.15 0.27

A total of 147 mesophilic Aeromonas strains and 30 Vibriostrains were tested. Vibrio strains showed affinity agglutina-tion patterns with the various coated latex beads (withconnective tissue and plasma proteins as well as glycopro-tein PAA reagents, but not with ovalbumin PAA, BSA PAA,or uncoated latex beads). These results indicated the pres-ence of specific cell surface receptors on various Vibriostrains for connective tissue proteins (Cn-I and Cn-IV, Fn,and Fg) and for sialic acid-rich glycoproteins (such as mucinsand orosomucoid). Yamamoto et al. (30, 31) reported thathemagglutinating V. cholerae 01 strains and V. choleraenon-O1 strains adhere to the human intestinal mucosa.Krovacek et al. (14) reported that A. hydrophila and Vibrioanguillarum adhere to fish mucus-coated, glass slides. Aero-monas and Vibrio strains isolated from diseased fish agglu-tinated bovine submaxillary mucin- and porcine stomachmucin-coated latex beads (Table 1).Aeromonas strains have a complex cell surface composed

of a mosaic of molecular structures which enable bacteria tobind to a range of biomolecules by lectinlike interactionsbetween adhesins and specific glycoconjugates. Several re-ports have demonstrated how Aeromonas strains bind tovarious erythrocytes (1), mouse adrenal cells (R. B. Clark, etal., Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B-191, p.50), fish cells, and cell surface glycoconjugates such as fishmucus (14).The Aeromonas strains used for the present study showed

a high tendency to agglutinate latex beads, coated anduncoated, and, in the case of the former, regardless of thetype of protein coating. It seems possible that nonspecificcharge or hydrophobic interactions participate in the PAAagglutinating reactions. The following observations led us tothis hypothesis. (i) A. hydrophila and related species (A.caviae and A. sobria) express high cell surface hydropho-bicity and charge as revealed by partitioning in aqueoustwo-phase systems of Dx and PEG (unpublished data). (ii)The various coated latex beads used were also hydrophobicand negatively charged (Table 5). (iii) Inhibition of aggluti-nation of latex beads coated with glycoproteins was obtained

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when the PAA was performed in the presence of 0.5%Tween 20 (Table 3).

Moreover, our results suggest that Aeromonas strainsexhibit surface structures capable of binding plasma glyco-proteins such as Fg, albumin, lactoferrin, hemocyanin, andhemolymph (F. Ascencio, A. Ljungh, and T. Wadstrom;unpublished data). It is probable that these binding charac-teristics are mediated by protein receptors, as has beendescribed for Streptococcus canis (15), and are the reasonwhy bacteria are able to agglutinate various PAA reagents.To rule out the possibility that nonspecific aggregations

contributed to PAA positivity, we also performed inhibitionassays (i.e., particle agglutination inhibition assays) (Table2). Agglutination reactions with Cn-I PAA and Fn PAAreagents were effectively blocked by incubating the bacterialsuspensions (of both Vibrio and Aeromonas strains) in thepresence of soluble gelatin. Further studies on inhibition ofVibrio and Aeromonas binding with natural and synthetic Cnand other proline-rich peptides (6) are now needed.

Bacterial agglutination of various glycoprotein- and mu-

cin-coated latex particles could be inhibited by specificglycoproteins or by monosaccharides (Table 3). Glycopro-teins (bovine fraction VI) and fetuin were the best inhibitors.

Hemagglutination inhibition with fetuin but not with singlemonosaccharides has been reported for a soluble hemagglu-tinin isolated from Aeromonas species (25); lack of suchinhibition with single monosaccharides may still be due tospecific interactions like that observed for the Escherichiacoli fimbria adhesin binding of disaccharides with galactose-a-1,4-galactose-type linkages (13).For Vibrio adhesins, the position of N-acetylneuraminic

acid in the glycoprotein carbohydrate structures, as well asthe galactose position, seems to be important for bacterialbinding to such complex sequences of carbohydrates. Infact, 60% of the Vibrio strains tested bound to beads coatedWith various types of mucins which contain sialic acid in ana-2,6 linkage; 44% of Vibrio strains bound to beads coatedwith orosomucoid, which has sialic acid in an ot-1,4 linkage,and 5% of all Vibrio strains bound to beads coated withfetuin, which has sialic acid in an a-2,3 linkage (Table 1). Theexact position of galactose in a glycoconjugate structure alsoseems to be important. Most (74%) of the Vibrio strainsbound to beads coated with asialofetuin, which has a P-1,4linkage; 19% of these strains bound to beads coated withdesialylated bovine submaxillary mucin, which has N-acetyl-D-galactosamine residues. Further studies are neededto elucidate the biochemical properties of the carbohydratereceptors of both Vibrio and Aeromonas species. We arenow studying the use of mucin- and glycoprotein-coatedlatex beads as PAA reagents to rapidly screen for bacterialsurface lectins.We found that many Vibrio strains bind '25I-labeled Fn

(data not shown) as reported by Wiersma et al. (29). Thenumber of Vibrio or Aeromonas strains that bind Fn PAAreagents corresponds to the strains that bind '251-labeled Fn,but the Fn PAA value does not correlate well with theamount of 125I-labeled protein binding. Similar test resultswere also obtained in Cn-I PAA and 125I-Cn-I binding assays

(data not shown). Lack of quantitative correlation betweenPAA and 125I-labeled protein binding has been reported (21).

Expression of bacterial cell surface properties commonlyvaries in relation to growth media and temperature (7, 24).Kabir and Ali (12) showed how the growth conditionsinfluenced the expression of cell surface hydrophobicity andhemagglutination properties of V. cholerae. Statner et al.(24) have reported growth effects on A. hydrophila cell

surface proteins. Other studies have shown that growthconditions influence the 125I-labeled protein binding to cellsof E. coli (A. Ljungh, L. Emody, H. Steinruch, B. Dahlback,P. Sullivan, E. Zetterberg, B. West, and T. Wadstrom,unpublished data) and various Salmonella strains (7). Pro-tein binding to Vibrio cells was significantly influenced bygrowth conditions (Table 4). For 125I-labeled Cn-I, the bestbinding conditions were obtained with cells grown on bloodagar at 25°C, while growth in peptone water at 25°C en-hanced 1251I-labeled Fg binding.

Studies on cell surface hydrophobicity and charge of bothVibrio and Aeromonas strains indicate that there is nocorrelation between agglutination of PAA reagents and cellsurface hydrophobicity or charge (data not shown).The classical hemagglutination assay is still commonly

used to screen for bacterial hemagglutinins or lectins (11, 25,27). However, latex particles coated with a single glycopro-tein or glycolipids with a-1,4 galactose-galactose disaccha-ride-type attachments (3) seem a more logical approach toscreen for bacterial lectins, especially for organisms such asVibrio and Aeromonas species able to produce a number ofvarious hemagglutinins (1, 11), and also because of the longstability of the PAA reagents compared with erythrocyteswhen stored at 4°C (21).With the promising PAA results (except for autoaggregat-

ing strains), we are now in the process of preparing humansmall bowel and fish mucin PAA reagents to test for thepresence of sugar-binding proteins (i.e., lectins) which mayserve as colonization factor antigens (adhesins) for initialcolonization of fish mucin and of the small bowel of humans(30).

ACKNOWLEDGMENTS

This study was supported by grants from The Swedish MedicalResearch Council (MFR 16X 04723) and from The Swedish Boardfor Technical Development with a scholarship to F.A.

LITERATURE CITED1. Atkinson, H. M., and T. J. Trust. 1980. Hemagglutinating

properties and adherence abilities of Aeromonas hydrophila.Infect. Immun. 27:938-946.

2. de Man, P., B. Cedergren, S. Enerback, A.-C. Larsson, H.Leffler, A.-L. Lundell, B. Nilsson, and C. Svanborg-Eden. 1987.Receptor-specific agglutination tests for detection of bacteriathat bind globoseries glycolipids. J. Clin. Microbiol. 25:401-406.

3. Essers, L., and K. Radebold. 1980. Rapid and reliable identifi-cation of Staphylococcus aureus by latex agglutination test. J.Clin. Microbiol. 12:641-643.

4. Faris, A., M. Lindahl, and T. Wadstrom. 1982. High surfacehydrophobicity of hemagglutinating Vibrio cholerae and othervibrios. Curr. Microbiol. 7:357-362.

5. Froman, G., L. M. Switalski, A. Faris, T. Wadstrom, and M.Hook. 1984. Binding of Escherichia coli to fibronectin. J. Biol.Chem. 23:14899-14905.

6. Gibbons, R. J., and D. I. Hay. 1988. Adsorbed salivary proline-rich proteins as bacterial receptors on apatitic surfaces, p.143-163. In L. Switalski et al. (ed.), Molecular mechanisms ofmicrobial adhesion. Springer-Verlag, New York.

7. Gonzalez, E. A., S. B. Baloda, J. Blanco, and T. Wadstrom.1988. Growth conditions for the expression of fibronectin andcollagen binding to Salmonella. Zentralbl. Bakteriol. Para-sitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 269:437-446.

8. Hazen, T. C., G. W. Esch, R. V. Dimock, and A. Mansfield.1982. Chemotaxis of Aeromonas hydrophila to the surfacemucus of fish. Curr. Microbiol. 7:371-375.

9. Isaacs, R. D., S. D. Paviour, D. E. Bunker, and S. D. R. Lang.1988. Wound infection with aerogenic Aeromonas strains: areview of twenty-seven cases. Eur. J. Clin. Microbiol. Infect.Dis. 7:355-360.

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VOL. 56, 1990

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