Journal of Applied Bacteriology 1993, 74, 149-154

Detection of Aerornonas hydrophila in food with an enzyme-linked immunosorbent assay

Susana Merino, Silvia Camprubi and J.M. Tomes Departamento de Microbiologia , Universidad de Barcelona, Barcelona, Spain

4104/02/92: accepted 4 August 1992

S. MERINO, s. CAMPRUBi AND J.M. TOMAS. 1993. A microtitration plate, antibody capture, enzyme-linked immunosorbent assay was developed for the detection of Aeromonas hydrophila serotype 0 : 11 (highly virulent strains). The assay utilizes a detector antibody which shows no cross-reactions with Aeromonas strains other than serotype 0 : 11 or non-Aeromonas competing organisms. T h e detector antibody is mixed with the sample and incubated for 1 h, microcentrifuged and the supernatant fluid (unadsorbed antibody) titred in a microtitre plate coated with A. hydrophila cells from serotype 0 : 11. All the A. hydrophila strains from serotype 0 : 11 tested reacted strongly with the detector antibody. Also by culturing and performing the immunoassay with the detector antibody we established and quantified the presence of A. hydrophila 0 : 11 in different foods.

INTRODUCTION

Mesophilic aeromonads are increasingly being reported as important pathogens of humans and lower vertebrates including amphibia, reptiles and fish (Janda 1987). Infec- tions produced by mesophilic aeromonads in humans can be classified into two major groups, i.e. non-invasive 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 lipopolysaccharide (LPS), permit classification of Aeromonus hydrophila into different categories on the basis of their virulence (Dooley et al. 1985; Janda et al. 1987).

Recently, a group of highly virulent A. hydrophila strains isolated from humans and fish has been described (Paula et al. 1988), serologically related to their 0-antigen LPS (serotype 0 : 11) and having a surface array protein of molecular weight 52 kDa (termed S-layer) (Dooley and Trust 1988). We obtained specific antibody against purified S-layer and used it as 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 foods, and we also describe a protocol for the quantification of these strains per gram of food.

MATERIALS AND METHODS

Bacterial strains and media

We have already used A . hydrophila strains TF7, L L l and ATCC 9071 from serotype 0 : 11 (Merino et al . 1990b).

Correspondenie l o : J . M . Tomas, Deparramenro de Microbrologia, llniriprsidad de Barcelona, DiaRonal6J5, OX071 Barrelona, Spain

Aeromonas hydrophila AH-38, AH-39, AH-40, AH-41, AH-42, AH-43 and AH-44 (serotype 0 : 11) were a gift from J.M. Janda (University of Berkeley, USA). Aeromonas hydrophila strains AH-3 and Ba5 (serotype 0 : 34), AH-50 (serotype 0 : 22), 24830, 24963, 25616, ATCC 7966, and A . salmonicida A-450 were also previously used by us (Merino et al. 1990a). Vibrio anguillarum serotype 0 1 and 02 , V. ordalii and V. parahaemolyticus were a gift from T. J. Trust (University of Victoria, Canada). Escherichia coli CSH57, Enterobacter agglomerans ATCC 2321 6, Ent. cloacae ATCC 12666, Klebsiella pneumonia C3, Salmonella typhimurium LT2, Serratia marcescens, Proteus vulgaris, and Yersinia enterocolitica ATCC 96 10 were previously used by us (Tomis et al. 1986).

The strains were usually cultured and maintained on tryptic soy broth (TSB) (Adsa, Barcelona, Spain) ; tryptic soy agar (TSA) was obtained by adding 1.5% agar. The growth temperature was usually 37°C for Enterobacte- riaceae and 28°C for Vibrionaceae.

Isolation of S-layer

The method of Dooley and Trust (1988) was used. Briefly, cells of A. hydrophila strains from serotype 0 : 11 were har- vested after growing for 24 h in TSB, washed three times in 20 mmol 1 ' Tris (pH 8), resuspended in 0.2 moll - ' glycine (pH 4) and stirred at 4°C for 15 rnin. The cells were removed by three sequential centrifugations at 12 000 g for 20 min at 4°C. The S-layer sheet material was col- lected by centrifugation at 40000 g for 30 min and washed once in Tris buffer. Protein concentrations were deter- mined by the Lowry et al. (1951) procedure with bovine serum albumin (Sigma) as the standard. Purified S-layer

150 SUSANA MERINO ET A L .

was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by a modification (Ames el al. 1973) of the Laemmli (1970) procedure. Protein gels were routinely stained with Coomassie blue.

Production of polyclonal antibodles

Polyclonal antiserum was raised in adult New Zealand white rabbits against purified S-layer (0.5 mg per dose). After being injected with three doses at 2-week intervals, the blood was collected from the marginal ear vein after 10 d. After centrifugation of blood samples the serum was col- lected and stored at - 20°C until used.

Western blotting

After SDS-PAGE of purified S-layer preparations or whole cells, the gel was equilibrated in transfer buffer (25 mmol 1- Tris, 192 mmol 1- glycine, 20% methanol) for 1 h. Proteins were transferred electrophoretically to nitro- cellulose paper (100 mV) for 1 h in a Mini-Transblot apparatus (BioRad). After blocking non-specific binding sites with phosphate-buffered saline (PBS)-1 YO bovine serum albumin overnight the nitrocellulose paper was incu- bated in a 1 : 250 dilution of anti-S-layer antibody for 2 h. The blot was washed several times in PBS-Tween and then incubated with alkaline phosphatase-conjugated goat anti- rabbit IgG (1 : 500 dilution) (Boehringer). The blot was developed with bromochloroindolyl phosphate-nitro blue tetrazolium (Boehringer) for 2 min. The reaction was stopped with PBS-20 mmol I - ' EDTA (Sigma), and the blot was air dried.

ELISA protocol

Cellcoated plates. Suspensions of A. hydrophila TF7 (serotype O : 11) cells (200 pI of 10 pg of cell protein ml - ') in phosphate buffer (0.1 mol I - ' ; pH 8) were placed in 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 tem- perature the solution was removed and the plates washed three times with water and left to dry in air, before storage in the dark at room temperature in sealed plastic bags con- taining silica gel as desiccant. Such plates showed a mean life of a t least 3 months.

Treatment of samples. The samples (10 g of raw seafood (oysters, clams or mussels) or raw fish (cod or sea-bass)) were homogenized with 90 ml of peptone water and left to incubate a t 28°C for 6 h ; 10 ml were then added to 90 ml of TSB and incubated at 28°C for 16 h. The culture was centrifuged and the cells resuspended in a small volume of the antibody solution (200 p l ) .

Detection of A. hydrophila 0: 7 7 . For the detection of A. hydrophila 0 : 11, treated samples or standard cell sus- pensions in PBS were mixed with specific detector antibody and incubated for 1 h at 37°C. After the adsorption time the mixture was centrifuged and the supernatant fluid (unadsorbed antibody) was added to the microtitre plate and incubated for 1 h at 37°C. After the reaction time, the plate was washed three times with PBS-Tween (5"% Tween 20), and incubated for 1 h at 37°C with a 1 : 2000 dilution in PBS-Tween of alkaline phosphatase-labelled goat anti- rabbit immunoglobulin G (Boehringer). After three washes with PBS-Tween, p-nitrophenyl phosphate (Boehringer), disodium salt, at 1 mg ml-' in 50 mmol 1- I 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.

Effect of high numbers of competing organisms

Tryptic soy broth (100 ml) was inoculated with A. hydro- phila 0 : 1 1 strains at approximately 1 cell per ml; to the same broths Aeromonas strains not belonging to serotypc 0 : 11 or other Vibrionaceae or Enterobacteriaceae were added at ratios from 1 : 1 to 1 : lo6. The broths were incu- bated for 16 h and sampled for the ELISA, as previously described for the food samples.

Detectlon of low numbers in food

Samples of sequentially filter-sterilized peptone water- seafood homogenate were inoculated with A. hydrophila 0 : 1 1 cells (from 10 to 1000 cells per 100 ml), and imme- diately sampled for the ELISA, as described above.

Quantification protocol

After filtration of 100 ml of TSB plus peptone water sample without incubation, the filter was placed on the surface of a TSA plate and incubated at 28°C for 16 h. After incubation the filter was air dried and blocked with 1% bovine serum albumin in PBS, washed with PBS- Tween and incubated with the detector antibody (1 : 100 dilution in PBS) for 2 h at room temperature. After the incubation with the first antibody the filter was washed twice with PBS and incubated with 1 : 1000 dilution of alk- aline phosphatase-labelled goat anti-rabbit immunoglobulin G (Boehringer) for 1 h at room temperature. Finally, 50 pg ml ' of 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer) in 0.1 mol I - ' Tris (pH 9.9, 0.1 mol 1- NaCl and 0.05 mol I - ' MgCl, were added. The positive colonies gave a dark violet colour. The number of positive colonies was the number of A. hydrophila 0 : 1 1 cells per gram of food sample.

DETECTION OF AEROMONAS HYDROPHILA 151

Fig. 2 Western-blot of whole cells from Aeromonas hydrophila 0 : 11 strains. Whole cells were submitted to SDS-PAGE and electroblotted as described in Materials and Methods and treated with specific anti-S-layer antibodies. Lanes: 1, strain TF7; 2, strain LLl, 3, strain ATCC 9071; 4, strain AH-38; 5, strain AH-39; 6, strain AH-40; 7, strain AH-41; 8, strain AH-42; 9, strain AH43 ; and 10, strain AH-44

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 strains (serotype 0 : 11 or other serotypes), other Vibrionaceae or different Enterobacteriaceae. (An example of this is shown

Fig. 1 SDS-PAGE of purified S-layers from Aeromonas hydrophila 0 : 11 strains obtained as described in Materials and Methods. Lanes: 1, strain TF7; 2, strain LLl; 3, strain AH-38; 4, strain AH-39; and 5, molecular size standards (14.0, 20.1, 30.0, 43.0,67.0 and 94.0 kDa from Pharmacia Fine Chemicals)

RESULTS

S-protein characterization

Figure 1 shows the protein profiles obtained by SDS- PAGE of purified S-layers from different A. hydrophila strains serotype 0 : 11. It is clear that we obtained only a single band (S-protein) of molecular weight ranging from 49 to 52 kDa depending on the strains.

Antibody characterization

We obtained antibodies against purified S-layer from strain TF7 (0 : 11) and tested them against different A. hydro- phila strains. Figure 2 shows that antibodies against puri- fied S-layer from strain TF7 reacted against whole cells of different strains of A. hydrophila serotype 0 : 11, rendering a strong band by Western-blotting which corresponds to a molecular weight also in the range 49-52 kDa (S-protein). Finally, Fig. 3 shows that A. hydrophila cells from non- 0 : 11 serotype (results are shown for A. hydrophila AH-3; serotype 0 : 34) did not react by Western-blotting with antibodies against purified S-layer from strain TF7.

Antibody cross-reactions

The cross-reactions of the antibody were studied by strutting dilution obtained were compared with the curve on a plate coated,

Fig. 3 Western-blot of whole cells from Aeromonas hydrophila using specific anti-S-layer antibodies. Lanes : A, strain TF7 (serotype 0 : 11); and B, strain AH-3 (serotype 0 : 34)

on cell-coated plates. The

152 SUSANA MERINO ET A L .

I00

A TI-.-.-. I serotyped but always lacking the S-layer and not highly virulent (Merino et a l . 1990a) showed, at least, a 100-fold decrease in the degree of cross-reaction in comparison with the A. hydrophila strains from serotype 0 : 11. Similar results were obtained with whole cells of V. anguillarum serotype 01 and 0 2 , V. ordalii, V . parahaemolyticus, and different Enterobacteriaceae (E. coli, Kl. pneumoniae, Ser . marcescens, Ent. cloacae, Ent. agglomerans, P r . vulgaris, Salm. typhimurium and Y . enterocolitica).

01 I I I , , , , , I , I I I 1 1 1 1 1 I I I I I I

I 10 I00 I000 I/ontiserum dilution x 100

Fig. 4 ELISA with different Aeromonas hydrophila strains. Antiserum (antibody against S-layer) was adsorbed separately with cells of TF7 (+) or LL1 (*) (serotype 0 : 1 1 ) or AH-3 (m) (serotype 0 : 34), and the unadsorbed antiserum was titred on cell-coated plates

in Fig. 4 for A. hydrophila strains TF7 and LL1 (serotype 0 : 11) and AH-3 (serotype 0 : 34).) The per cent cross reaction was calculated by dividing the A,,, obtained with the unadsorbed antibody by the A405 obtained with the antibody adsorbed with whole cells. The results obtained in this manner are given in Table 1.

The detector antibody shows a high degree of cross- reaction only with A. hydrophila strains belonging to serotype 0 : 11 or the purified S-layer from these cells. Other Aeromonas strains from different serotypes or not

ELISA standard curves

Standard curves obtained for different amounts of A . hydrophila 0 : 11 cells added for adsorption with specific antiserum (1 : 500 dilution) are shown in Fig. 5 . The stan- dard curves show that the assay has a very low limit of detection, being able to pick up as few as 10 cells per 100 ml of peptone water sample. This is due to the high speci- ficity of the antibody, and also because the antigen used (S- layer) is a unique structure from A. h,ydrophila strains serotype 0 : 11 (Dooley and Trust 1988). A similar struc- ture named A-layer from A. salmoniczdu (Kay et al. 1981) is unable to cross react with this antiserum (Table 1). With all these results we defined a positive ELISA when the A405 of the sample was inferior to 80% of the A,,, of the unadsorbed antibody ( lo5 cells of non-A. hydrophila 0 : 11 per ml are completely negative and 10' cells of A. hydro- phila 0 : 11 per ml are positive, see Figs 4 and 5).

Table 1 Cross-reactions of polyclonal antiserum against S-layer* with different whole cells

Yo Yo Cross-reactant Cross-reaction Cross-reactant Cross-reaction

S-iayer LL 1 AH-38 AH-40 AH-42 AH-44 AH-3 AH-50 24963 ATCC 7966 Vibrio anguillarum 01

ordabi Escherichra coli Klebsrella pneumoniae Enlerobacrer agglomerans Proreus vulgaris

20.6 20.5 20.4 20.3 20.4 20.4

0.15 0.12 0.12 0.09 0.06 0.04 0.03 0-04 0.05 0.03

TF7 ATCC 9071 AH-39

AH43 AH-41

Ba 5 24830 25616 Aeromonas salmonicida A-450 Vibrio anguillarum 0 2

parahaemolyticus Salmonella typhimurium Enterobacter cloacae Serraria marcescens Yersinia enterocolitica

20.5 20.4 20.5 20.5 20.2

0.14 0.13 0.08 0.05 0.06 0.07 0.04 0.04 0.05 0.03

* Data were obtained by comparing antibody binding to cell-coated plates. The values are the average of three independent experiments. Standard deviations were always less than 0.03. Strains TF7, LLI, ATCC 9071, AH-38, AH-39, AH-40, AH-41, AH-42, AH-43 and AH44 are A . hydrophila 0 : 11 (S-layer'). Strains AH-3, Ba5, AH-SO, 24830, 24963, 25616 and ATCC 7966 are A . hydrophila not belonging to serotype 0 : 1 1 (S-layer-).

DETECTION OF AEROMONAS HYDROPHILA 153

Cells mi-'

Flg. 5 Standard curve for different amounts of Aeromonas hydrophila cells of T F 7 ( +) (serotype 0 : 11) or AH-3 (m) (serotype 0 : 34) used for the adsorption of the specific antiserum used at 1/500 dilution. The unadsorbed antiserum was assayed on cell-coated plates

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 sterilized peptone water-seafood sample (the seafood samples in this case were mussels obtained from seafood farms; Delta del Ebre, Catalunya, Spain) inoculated with a small number of A . hydrophila 0 : 1 1 cells and varying numbers of non-A. hydrophila 0 : 1 1 cells. Table 2 shows that all samples inoculated with 1.4 cells ml-' of A. hydro- phila TF7 (serotype 0 : 1 1 ) were positive, even in the pres- ence of a large inoculum (lo6 cells ml- ') of different non-A. hydrophila 0 : 1 1 competing cells.

These results prompted us to examine the possibility of quantifying the assay in order to establish the original numbers of these cells present in a food sample. As shown in Table 3, there is a good correlation between the number of A. hydrophila TF7 cells (serotype 0 : 1 1 ) inoculated and

Table 2 Detection of Aeromonas hydrophila 0 : 11 in the presence of

the number of positive colonies found in the quantified assay, independent of the inoculum and the kind of food sample (with low or high numbers of Aeromonas spp. as a measure of the degree of contamination). Some positive colonies (more than 100) were identified as A. hydrophila serotype 0 : 1 1 by API 20NE (as recommended by Analytabs), oxidase, DNase, resistance to 0/129 agent, motility and the ability to autoagglutinate which is unique for this serotype (Paula et al . 1988). The number of positive colonies reconfirmed was 99%. None of the negative colo- nies tested (more than 50) were identified as A. hydrophila serotype 0 : 1 1 .

DISCUSSION

This study has shown the potential advantages of using a specific immunoassay for the detection of highly virulent A. hydrophila (strains from serotype 0 : 1 1 ) in food. The fact that only A . hydrophila serotype 0 : 1 1 strains are able to produce S-layer Uanda et al. 1987), and the production of antibodies against this unique structure, allowed us to design an easy and highly specific immunoassay for detect- ing these strains. The specificity of the assay is such (see Table 1) that there is no need for selective culture and only one incubation step is necessary to allow damaged cells to recover and start multiplying.

The specificity of this assay is also illustrated by the study in Table 2, in which small numbers of A. hydrophila serotype 0 : 1 1 cells were inoculated with increasing numbers of A. hydrophila serotypes 0 : 34 and 0 : 22, V . anguillarum, E. coli, and Kl. pneumoniae cells. As few as 1.4 A. hydrophila serotype 0 : 1 1 cells ml - I were still detect- able by the antibody assay (as can also be observed in the standard curves shown in Fig. 2), despite viable counts of lo6 cells ml-' of the other bacteria. This work shows that there is no cross-reaction with either of these organisms at high levels, confirming the lack of cross-reactivity shown in Table 1 , and also the absence of other effects such as physi- cal interference that might be expected at these levels. The

No. of cells ml- of inoculum

increasing numbers of competing organisms Vibrio Escherichia Klebsiella

A . hjidrophila

0 : 11 0 : 34 0 : 22 anguillarum coli pneumoniae ELISA

- - - - + 1.4 1.4 3.2 2.7 3.1 2.8 5.2 + 1.4 2x10' 3x10 ' 2x10 ' 4 x 10' 3 x lo2 + 1.4 8x104 3x104 4x104 5 x 104 2 x 104 + 1.4 6x106 2x106 3x106 5 x 106 4 x lo6 + - 6x106 2x106 3x106 5 x 106 4 x lo6 -

-

+ and - ELISA results are as previously defined in the Results.

154 SUSANA MERINO E T A L .

Table 3 Detection of Aeromonas hydrophila 0 : 1 1 by ELISA and the quantification protocol after inoculation of different tryptic soy broth (TSB)-peptone water samples with A . hydrophila TF7 cells (serotype 0 : 11)

ACKNOWLEDGEMENTS

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

Aeromonas spp. Inoculum viable count Quantification

Sample 100 ml-'* 100 ml- ' t ELISA protocol

1 1 1 1 2 2 2 3 3 3 4 4

0 11

198 988

0 129 53 1

0 15

1 I7 0

123

5.2 x 10'

6.9 x 10' 1.6 x 10' 8.4 x lo6 8.3 x lo6 8.5 x lo6

5.4 x 102

4.8 x 103 4.8 x 103 4.9 x 103

3.6 x 10' 3.7 x 104

O$ - + 10 + 189 + 972

0 + 122 + 519

0 + 14 + 112

0 + 117

-

-

-

* Inoculum per 100 ml of TSB plus peptone water from the dif- ferent samples. Sample 1, raw clams; sample 2, raw mussels; sample 3, raw oysters; and sample 4, raw sea-bass. t Aeromonas spp. viable counts were performed by plating on tryptic soy agar plus 30 pg of ampicillin and 5% sheep blood, as previously reported (Mishra et al. 1987). 1 Some positive and negative colonies were reconfirmed as described in the Results. + and - ELISA results are as previously defined in the Results.

main reason that these effects cannot be seen is the fact that the antigen used (Slayer) for the antiserum production is a unique structure from A . hydrophila strains serotype 0 : 11 (Dooley and Trus t 1988).

T h e combination of sensitivity and specificity provided in this ELISA resulted in a very rapid assay in which low numbers of highly virulent A . hydrophila cells (serotype 0 : 11) can be detected in as little as 24 h. T h e studies in food (Table 3) show that this immunoassay is largely unaf- fected by the food matrix, as well as by the number of other bacteria contained in the food (quantified in our assay as the total number of Aeromonas spp. in the sample food). I t is also clear that we can quantify the immunoassay, because there is a good correlation between the number of TF7 cells (serotype 0 : 11) added to the food sample and the number of positive colonies found in the immunoassay. Also, the quantified assay is unaffected by the food matrix or by the number of other bacteria contained in the food.

We hope that the feasibility of this immunoassay for detecting highly virulent A. hydrophila (serotype 0 : 11) in food will be useful in the formulation of microbiological quality standards, especially for fish and seafood as a conse- quence of the introduction of a new standard ( A . hydrophila 0 : 11) not previously determined.

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