Enzyme-Linked Immunosorbent Assay for Detection of Highly Virulent Strains of

Aeromonas hydrophilia and Aeromonas sobria in Water

SUSANA MERINO and SILVIA CAMPRUBI Departamento de Microbiologia, Universidad de Barcelona, Diagonal

645, 08071 Barcelona, Spain

MIGUEL REGUE Departamento de Microbiologia y Parasitologia Sanitarias,

Universidad de Barcelona, Diagonal sln, 08028 Barcelona, Spain

JUAN M. TOMAS" Departamento de Microbiologia, Universidad de Barcelona, Diagonal

645, 08071 Barcelona, Spain

ABSTRACT

A microtitration plate, antibody capture, enzyme-linked immunosorbent assay was devel- oped for detection of Aeromonas hydrophila and sobria serotype 0 : 11 (highly virulent strains). The assay utilizes a detector antibody that shows no cross-reactions with Aero- monas strains not belonging to serotype 0 : 11 or nonderomonas competing organisms. All the A. hydrophila and sobria strains from serotype 0 : 11 tested reacted strongly with the detector antibody. Also, by culturing and performing the immunoassay on a filter with the detector antibody, we can quantify A. hydrophilia or sobria 0 : 11 cellsI100 mL of water. 0 1993 John Wiley & Sons, Inc.

INTRODUCTION

Mesophilic aeromonads are increasingly being reported as important pathogens of humans and lower vertebrates, including amphibia, rep-

* To whom all correspondence should be addressed.

Environmental Toxicology and Water Quality: An International Journal Vol. 8,451-460 (1993) 0 1993 John Wiley & Sons, Inc. ccc 1053-4725/93/040451-10

452/MERINO ET AL.

tiles, and fish (Janda, 1987). Infections produced by mesophilic aeromo- nads in humans can be classified into two major groups-i.e., noninva- sive disease such as gastroenteritis and systemic illnesses (Janda and Brenden, 1987). Surface characteristics, such as the presence of an S- layer or the type of LPS, permit classification of Aeromonas hydrophila into different categories on the basis of their virulence (Janda et al., 1987; Dooley et al., 1985).

Recently, a group of highly virulent A. hydrophilia and sobria strains isolated from humans and fish have been described (Paula et al., 1988; Kokka et aZ., 19911, serologically related by their 0-antigen lipopolysaccharide (serotype 0 : 11) and having a surface array protein of molecular weight 52 Kda (termed S-layer) (Dooley and Trust, 1988). By obtaining specific antiserum against purified Slayer, we used this antibody as the detector antibody in an enzyme-linked immunosorbent assay (ELISA) developed by us. In this study we describe an ELISA for detection of this group of mesophilic aeromonads (serotype 0 : 11) in water, and we also describe a protocol for the quantification of these strains in 100 mL of water.

MATERIALS AND METHODS

Bacterial Strains and Media

Aeromonas hydrophila strains TF7 and LL1 from serotype 0 : 11 were previously used by us (Merino et al., 1990b). Aeromonas hydrophila AH-38, AH-39, AH-40, and AH-41 (serotype 0 : 11) were a gift from J.M. Janda (University of Berkeley, USA), as well as A. sobria ATCC9071 and 53 (rough) (Kokka et al., 1991). Aeromonas hydrophilia strains AH-3 and Ba5 (serotype 0:34), AH-50 (serotype 0:22), and 24830,24963,25616, and ATCC7966, were previously used by us (Me- rino et al., 1990a). Aeromonas salmonicida A-450 and A-440 (Kay et aZ., 19811, pathogenic strains for fish containing a Slayer (named A- layer), were a gift from W.W. Kay (University of Victoria, Canada). Vibrio anguillarum serotype 01 and 02, V. ordalii, and V. parahaemo- lyticus were from our lab stock. Escherichia coli CSH57, Enterobacter agglomerans ATCC23216, Enterobacter cloacae ATCC12666, Klebsiella pneumoniae C3, Salmonella typhimurium LT2, Serratia marcescens, Proteus vulgaris, and Yersinia enterocolitica ATCC9610 were pre- viously used by us (Tomas et al., 1986).

The strains were usually cultured and maintained on tryptic soy broth (TSB). TSB agar was obtained by adding 1.5% agar (TSA). The growth temperature was usually 37°C for Enterobacteriaceae and 20°C for Vibrionaceae.

ELISA DETECTION OF AEROMONASJ453

Isolation of S-Layer

We used the method of Dooley and Trust (1988). To briefly explain this procedure: Cells of A. hydrophila strains from serotype 0 : 11 (mainly strain TF7) were harvested after 24 h of growth in TSB, washed three times in 20 mM Tris (pH 8), resuspended in 0.2 M glycine (pH 4), and stirred at 4°C for 15 min. The cells were removed by three sequential centrifugations at 12 .000~ g for 20 min at 4°C. The S-layer sheet material was collected by centrifugation at 40.000X g for 30 min and washed once in Tris buffer.

Production of Polyclonal Antibodies

Polyclonal antiserum was raised in adult New Zealand white rabbits against purified S-layer (0.5 mg/dose). After being injected with three doses at two-week intervals, the blood was collected from the marginal ear vein after 10 days. After centrifugation of blood samples, the plasma was removed and stored at -20°C until used.

ELISA Protocol

Cell-Coated Plates

To prepare the plates, suspensions of A. hydrophila TF7 (serotype 0 : 11) cells (200 pL of 10 pg of cell protein mL-') in phosphate buffer (0.1 mol L-'; pH 8) were placed in all the 96-well microtitration plates. After 16 h at 4"C, the plates were washed three times with water before bovine serum albumin (200 pL of 10 mg mL-' in phosphate buffer pH 8) was added to block unreacted sites. After 2 h at room temperature, the solution was left and the plates washed three times with water and left to dry in air before being stored in the dark in sealed plastic bags containing silica gel as desiccant.

Treatment of Samples

Samples of 100 mL of water were filtered (0.22 p pore size) and the filter were incubated for 10 h in TSB. After the growth, the cells were centrifuged and resuspended with a small volume of the antibody solu- tion (200 pL).

Detection of A. hydrophila 0 : 11

For the detection of A. hydrophila 0 : 11, treated samples or standard cell suspensions in PBS were mixed with specific detector antibody

454/MERINO ET AL.

n t i s 20

-i 1-1 -LJ i l l I

1 10 100 1000

liantiserum dilution x 100 Fig. 1. ELISA with different A. hydrophila strains. Antiserum (antibody against

S-layer) was adsorbed separately with cells of TF7 (+) or LL1 (*I (serotype 0 : 111, or AH-3 (W) (serotype 0:34), and the unadsorbed antiserum was titered on cell-coated plates.

and incubated for 1 h at 37°C. After the adsorption, the mixture was centrifuged and the supernatant (unadsorbed antibody) was added to the microtiter plate and incubated for 1 h at 37°C. After the reaction time, the plate was washed three times and incubated for 1 h at 37°C with a 1 : 2000 dilution in PBS-Tween of alkaline phosphatase-labeled goat antirabbit immunoglobulin G (Boehringer). Finally, p-nitrophenyl phosphate, disodium salt, at 1 mg mL-' in 50 mM carbonate buffer (pH 9.6), was added and the A405 was read after incubation at 37°C for 30 min. Unabsorbed antibody or antibody adsorbed with whole cells of A. hydrophila strains from serotype 0 : 11 were used as controls. We defined a positive ELISA when the A,,, of the sample was inferior to the 80% of the unadsorbed antibody (lo5 cells of non A. hydrophila 0 : l l /mL are completely negative and lo1 cells of A. hydrophila or sobria 0 : l l /mL are positive; see Figs. 1 and 2).

Effect of High Numbers of Competing Organisms

TSB broths (100 mL) were inoculated with A. hydrophila 0 : 11 strains at approximately 1 cell mL-l; to the same broths were added Aeromonas strains not belonging to serotype 0 : 11 or other Vibrionaceae or Entero-

ELISA DETECTION OF AEROMONAS1455

%

f 0

U n a d 8 0 r b e d

a n t i

e r

rn

8

U

8o F 6ol

10 100 1000 10000 100000

cells/ml Fig. 2. Standard curve for different amounts of A. hydrophila cells of TF7 (+)

(serotype 0 : 11) or AH-3 (a) (serotype 0 : 34) used for the adsorption of the specific antiserum used a t 1/500 dilution. The unadsorbed antiserum was assayed on cell-coated plates. Standard deviations were always inferior to 1%.

bacteriaceae at ratios from 1 : 1 to 1 : lo6. The broths were incubated for 12 h and sampled for the ELISA, as previously described for water samples.

Detection of Low Numbers in Water

Samples of filtered water were inoculated with A. hydrophila 0 : 11 cells (from 5 to 1000 cells/100 mL) and immediately were sampled for the ELISA as previously described.

Quantification Protocol

After filtration of 100 mL of water, the filter was incubated onto a surface of a TSA plate for 14 h at 20°C. After the incubation period, the filter was air dried and blocked with 1% bovine serum albumin in PBS, washed with PBS-Tween, and incubated with the detector anti- body (1 : 100 dilution in PBS) for 2 h at room temperature. After the incubation with the first antibody, the filter were washed twice with PBS and incubated with 1 : 1000 dilution of alkaline phosphatase-la- beled goat antirabbit immunoglobulin G for 1 h at room temperature.

456/MERINO ET AL.

TABLE I Cross-reactions of polyclonal antiserum against S-layera with different whole cellsb

Cross-reactant % Cross-reaction Cross-reactant % Cross-reaction

S-layer 100 TF7 100 LL1 100 AH-38 99 AH-39 100 AH-40 99 AH-41 100 ATCC907 1 99 53 (rough) 0.8 AH-3 0.7 Ba5 0.6 AH-50 0.6 24830 0.6 24963 0.6 25616 0.4 ATCC7966 0.4 A. salmonicida A-450 0.3 A. salmonicida A-440 0.3 V. anguillarum 01 0.3 V. anguillarum 02 0.3 V. ordalii 0.2 V. parahaemolyticus 0.3 E . coli 0.2 S. typhimurium 0.2 K. pneumoniae 0.2 E . cloacae 0.2

P . uulgaris 0.2 Y . enterocolitica 0.2 E . agglomerans 0.3 S . marcescens 0.3

a Data were obtained by comparing antibody binding to cell-coated plates. The per- centage of TF7 cross-reaction was considered as reference (100%).

Strains TF7, LL1, AH-38, AH-39, AH-40, and AH-41 are A. hydrophila 0: 11 and ATCC9071 A. sobria 0 : 11 (S-layer'). Strain 53 is a rough strain ofA. sobria 0- (S-layert). Strains AH-3, Ba5, AH-50,24830,24963,25616, and ATCC7966 are A. hydrophila not belonging to serotype 0 : 11 (S-layer-). Aeromonas salmonicida A-450 and A-440 strains are Slayer+ [the S-layer with different monomeric protein (A-protein) than the S protein (monomeric protein of the S-layer ofA. hydrophila and sobria serotype 0: ll)].

Finally, 50 pg mL-l of 5-bromo-4-chloro-3-indolyl-phosphate in 0.1 M Tris (pH 9.5) 0.1 M NaCl 0.05 M MgCl, were added, and the positive colonies give a dark violet color while no color could be observed for the negative ones. The number of positive colonies is the number of A. hydrophila 0 : 11 cells (highly virulent)/100 mL of water sample.

RESULTS AND DISCUSSION

Antibody Cross-Reactions

The cross-reactions of the antibody were studied by constructing dilu- tion curves on cell-coated plates. The curves obtained were all compared with the curve on a plate coated, as previously described in Materials and Methods, with A. hydrophila TF7 cells (serotype 0 : 11) and the antibody adsorbed with different whole cells of A. hydrophila or sobria strains (serotype 0 : 11 or other serotypes), other Vibrionaceae, or differ- ent Enterobacteriaceae. (An example of this is shown in Fig. 1 for A.

ELISA DETECTION OF AEROMONASl457

hydrophila strains TF7 and LL1 (serotype 0 : 11) and AH-3 (serotype 0 : 34). The percent cross-reaction was calculated by dividing the A,,, obtained with the unadsorbed antibody by the A,,, obtained with the antibody adsorbed with whole cells. The percentage of TF7 cross-reac- tion was considered as reference (100%). The results obtained in this manner are given in Table I.

The detector antibody shows a high degree of cross-reaction only with A. hydrophila or sobria strains belonging to serotype 0 : 11 or the purified S-layer from these cells. Other Aeromonas strains from different serotypes or not serotyped but always lacking the S-layer and not highly virulent (Merino et al., 1990a; Merino et al., 1990b) showed, at least, 100-fold decrease in the degree of cross-reaction in comparison with the A. hydrophila strains from serotype 0 : 11. Similar results were obtained using whole cells of V. anguillarum serotype 01 and 02, V. ordalii, V . parahaemolyticus, and different Enterobacteriaceae (E . coli, K . pneumoniae, S . marcescens, E . cloacae, E . agglomerans, P. vul- garis, S . typhimurium, and Y . enterocolitica). From these results we can conclude that the detector antibody is specific for A. hydrophila and sobria strains serotype 0 : 11 (highly virulents).

ELISA Standard Curves

Standard curves obtained for different amounts of A. hydrophila 0 : 11 cells used on adsorption on specific antiserum (1:500 dilution) are shown in Fig. 2. The standard curves shown in Fig. 2 demonstrate that the assay has a very low limit of detection, being able to pick up as few as 10 cells/100 mL of water sample. This is due to the high specificity of the antibody, and also to the antigen used (the S protein), which is the unique protein of the S-layer from A. hydrophila and sobria strains serotype 0 : 11 (Dooley and Trust, 1988; Kokka et al., 1991). Besides a similar S-layer (named A-layer) is observed on A. salmonicida strains A-450 and A-440 (Kay et al., 19811, where the unique protein is the A protein, different from the S protein from A. hydrophila and sobria 0 : 11, no cross-reactivity was observed between A. salmonicida cells with our antiserum (Table I).

Detection of A. hydrophila Strains Serotype 0: 11 in the Presence of Large Numbers of Competing Organisms

The presence of large numbers of microbial cells could interfere in the assay in spite of antibody specificity. In order to test this point, we studied the capacity of the assay in a filtered water sample (the water samples were obtained from different river sources near the Barcelona

4581MERINO ET AL.

TABLE I1 Detection of A . hydrophila and sobria 0: 11 in the presence of increasing numbers of

competing organisms

N" of cells ml of inoculum-'

A . hydrophila Vibrio Klebsiella 0 : l l 0:34 0 : 22 anguillarum E. coli pneumoniae ELISA

2.1 - 2.1 3.2 2.7 3.1 2.8 5.2 + 2.1 2 x 102 3 x 102 2 x 102 4 x 102 3 x 102 + 2.1 8 X lo4 3 X lo4 4 x 104 5 x 104 2 x 104 + 2.1 6 X lo6 2 X lo6 3 x 106 5 x 106 4 x 106 + - 6 x lo6 2 x lo6 3 x 106 5 x 106 4 x 106 -

+ - - - -

area, Spain) inoculated with a small amount of cells of A. hydrophila 0 : 11 and different amounts of non-A. hydrophila 0 : 11 cells. As can be observed in Table 11, all samples inoculated with 2.1 cells mL-l of A. hydrophila TF7 (serotype 0 : 11) were positive, even in the presence of a vast inoculum (lo6 cells mL-l) of different non-A. hydrophila 0:11 competing cells.

The combination of sensitivity and specificity provided in this

TABLE I11 Detection of A . hydrophila and sobria 0 : 11 by ELISA and the quantification protocol after inoculation of different water samples with A. hydrophila TF7 cells (serotype 0 : 11)

A . hydrophila E . coli viable count viable count ELISA Quantification

Sample 100 rnL-la 100 mL-lb result protocol

1 1 1 1 1 1 2 2 2 3 3 3

0 11 36

198 49 1 988

0 129 53 1

0 15

117

5 x 102 5 x 102 5 x 102 5 x 102 5 x 102 5 x 102 8 X lo6 8 x 106 8 x lo6 4 x 103 4 x 103 4 x 103

-

+ + + + + -

+ + -

+ +

0 10 34

189 487 972

0 122 519

0 14

112

a Aeromonas hydrophila 0 : 11 viable count were performed by plating on TSA plus 30 yg of ampicillin and 5% of sheep blood, as previously reported (Mishra et al., 1987).

Escherichio coli viable count were performed by plating on EMB agar (American Public Health Association. 1975).

ELISA DETECTION OF AEROMONASI469

ELISA resulted in a very rapid assay in which low numbers of A. hydrophila and sobria 0 : 11 cells (highly virulent) can be detected in as little as 20 h. These results prompted us to examine whether we could quantify the assay in order to establish the original numbers of these cells present in a water sample. As shown in Table 111, there is good correlation between the number of viable cells and the number of positive colonies found in the quantified assay, independent of the inoculum and the kind of water sample (with low or high numbers of E. coli as a measure of the contamination degree).

We hope that the feasibility of this immunoassay for detecting A. hydrophila and sobria, highly virulent (serotype 0 : 11) in water, would be of interest in order to enhance the microbiological water quality in different countries, as consequence of the introduction of a new stan- dard (A. hydrophila and sobria 0 : 11) not previously determined.

Part of this work has been supported by a PETRI grant (PTR89-0129) from Ministerio de Educaci6n y Ciencia.

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