Appl Microbiol Biotechnol (1990) 34:242-247

ApplieA Microbiology Biotechnology © Springer-Verlag1990

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods Bahram Mirhabibollahi, Joy L. Brooks, and Rohan G. Kroll Department of Microbiology, AFRC Institute of Food Research, Reading Laboratory, Shinfield, Reading, Berkshire, RG2 9AT, UK Received 16 March 1990/Accepted 28 June 1990

Summary. A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at c a . 10 4 cfu/ml, and at ca. 105 cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperometric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.

Introduction Traditional microbiological methods of detecting and enumerating contaminating microorganisms in foods are labour-intensive, cumbersome and slow for results to be obtained. Methods for obtaining rapid results equivalent to total viable counts based on assays of microbial ATP (La Rocco et al. 1986; Webster et al. 1988) or epifluorescent microscopy (Pettipher and Rodrigues 1982; Rodrigues and Kroll 1988) have been successfully developed. Genus- or species-specific methods for food-borne bacterial pathogens have also been developed, based on immunological (Mattingly 1984; Beumer and Brinkman 1989) or nucleic acid hybridisation probe (Fitts et al. 1983; Klinger et al. 1988) specificity. •The immunological methods, usually in the form of enzyme linked immunosorbent assays (ELISA), have,

Offprint requests to: R. G. Kroll

so far, received the most attention for practical application (Morris 1985). These ELISA methods can be fairly rapid (3 h) and specific, but they are insensitive (requiring > 106 target organisms/ml) and food samples have to be incubated prior to analysis to increase the number of target organisms to detectable levels. A major disadvantage is that every step in the assay requires a rigorous washing procedure, (i.e. the assay is heterogeneous), which is labour-intensive and difficult to automate. Attempts have been made to develop more simple immunoassays. Solid-phase immunofiltration (Ijsselmuiden et al. 1989) and homogeneous colorimetric enzyme immunoassays have been described (Gibbons et al. 1985; Kabanov et al. 1989) in which there are no washing steps. Furthermore, new types of amperometric and potentiometric homogeneous immunosensors have been described which operate on electrochemical detection principles (Di Gleria et al. 1986; Kjellstrrm and Bachas 1989). The main advantages of homogeneous assays are simplicity, ease of operation and, with the direct electrical signals produced by immunosensors, the possibility of automated methods and easy data processing. Automated electrical methods have been used in food microbiology for many years (Firstenberg-Eden 1986). These are based on the detection of changes in the conductance and/or impedance of the suspending medium. However, results are not obtained rapidly and the methods are not entirely specific. We have described a prototype immunosensor for the enumeration of protein A-bearing Staphylococcus aureus involving a direct, heterogeneous, catalase-based sandwich ELISA to protein A with an oxygen electrode detection step (Mirhabibollahi et al. 1990a). Although quite sensitive (ca. 10 4 cfu/ml), the electrical detection procedure for each sample was slow and technically difficult to perform. The assay used antibody-coated membranes in glass containers and therefore was not easily liable to automation. Furthermore, there was an unexplicable variation in the background values between different strains of S. aureus.

243 D o y l e et al. (1984) d e v e l o p e d a n i m m u n o s e n s o r using a l k a l i n e - p h o s p h a t a s e - l a b e l l e d a n t i b o d y , p h e n y l p h o s p h a t e as t h e s u b s t r a t e a n d e l e c t r o c h e m i c a l d e t e c tion of the product (phenol) by a carbon plate electrode i n c o r p o r a t i n g a l i q u i d c h r o m a t o g r a p h y s e p a r a t i o n . This s y s t e m was v e r y sensitive a n d c o u l d d e t e c t 1 n g / m l o f human orosmucoid glycoprotein; on the other hand, t h e results w e r e n o t q u i c k l y a v a i l a b l e ( > 12 h). A similar procedure using a sandwich assay, incorporating a s e p a r a t i o n step o n a n o c t y l d e c a s i l a n e c o l u m n ( W e h m e y e r et al. 1985) c o u l d d e t e c t r a b b i t i m m u n o g l o b u l i n ( I g G ) at 10 ng/1. This s y s t e m c o u l d b e u s e d to d e t e c t S. a u r e u s u s i n g a n a n o d i c p l a t i n u m e l e c t r o d e to d e t e c t t h e p h e n o l d i r e c t l y ( M i r h a b i b o l l a h i et al. 1990b). T h i s ass a y was q u i t e sensitive (ca. 103 c f u / g ) a n d the v a r i a t i o n in b a c k g r o u n d levels b e t w e e n strains o f S . a u r e u s was r e d u c e d . H o w e v e r , this i m m u n o s e n s o r a s s a y i n v o l v e d s e v e r a l r i g o r o u s w a s h i n g steps w h i c h m a d e it l a b o u r i n t e n s i v e a n d n o t e a s i l y c o n v e r t i b l e to a u t o m a t i o n . T h e f o o d i n d u s t r y r e q u i r e s r a p i d a n d sensitive m e t h o d s t h a t a r e l a b o u r s a v i n g a n d s i m p l e to p e r f o r m for detecting food-borne pathogens. We have adapted a n d i n v e s t i g a t e d the use o f the a l k a l i n e p h o s p h a t a s e / , p h e n o l / p l a t i n u m e l e c t r o d e system in a s e m i - h o m o g e n e o u s e n z y m e - l i n k e d a m p e r o m e t r i c i m m u n o s e n s o r syst e m to d e t e c t a n d e n u m e r a t e p r o t e i n A - b e a r i n g S . aureus b o t h in p u r e c u l t u r e s a n d in f o o d s . T h e results are d i s c u s s e d w i t h r e s p e c t to t h e p o t e n t i a l o f this t e c h n i q u e for automatic and reliable enumeration of other foodborne pathogens.

Materials and methods

All chemicals were of analytical grade from BDH, Poole, Dorset, UK, except staphylococcal protein A, phenylphosphate (disodium salt), potassium chloride, Tween 20 and the immunological reagents which were from Sigma, Poole, Dorset, UtC Microtitre plates (96-well) were from Western Laboratory Services (Aldershot, Hants, UK) and stomacher bags from Seward Medical (London, UK). Bacterial cultures and food samples. Pure cultures of S. aureus

NCDO 949, 1022, 1499 and 2044 were maintained on slopes of solidified Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, Hants, UK) by monthly subculture and storage at 4 ° C. Broth cultures were obtained by inoculating 20 ml BHI broth with a loopful of culture and incubating for 18 h at 37°C. Plate counts were performed by serial dilution of experimental broth cultures or food samples in sterile phosphate buffered saline (PBS, pH 7.3) and surface plating (0.1 ml) of the appropriate dilutions on BairdParker Agar (Oxoid) followed by incubation of the plates at 37 ° C for 2 days. Broth cultures for use as test antigen were diluted in PBST (PBS containing 0.1% v/v Tween 20) containing 1% w/v non-fat dried skimmed milk (PBSTM). Samples of semi-skimmed pasteurised milk (10 ml), Cheddar cheese (10 g), chicken (10 g) or braising beef (10 g) were inoculated with either pure cultures of S. aureus NCDO 949 or a mixture of the four NCDO strains of S. aureus. Meat and cheese samples were homogenised with 90 ml PBS in stomacher bags and diluted using homogenates of uncontaminated meat or cheese samples. Milk samples were diluted with non-inoculated milk to give food samples containing a range of cell densities of S. aureus cells. In some experiments samples (10 g or I0 ml) of beef, milk or chicken were inoculated with approximately 1-2 cfu/g or/ml of S.

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F~g. 1. Schematic representation of the immunoassay procedure. After adsorption of human immunoglobulin G (IgG) to the microtitre plate wells (sta~e ~, the antigen (A~), secondaw antibody (2~Ab) and anti-2~Ab alkaline phosphatase conjugate (AP.Ab) were added sequentially and incubated at room temperature for a total of 3 h. At the end of sta~e II, the microtitre plate wells were washed with phosphate-buffered saline containing 0.1% Tween 20 and the substrate, phenyl phosphate (PhP) added. Mter incubation for 1 h at 37 ° C, a sample (190 ~1) was removed and the product, phenol (Ph) detected in a separate electrochemical cell aureus NCDO 949. The inoculated samples were incubated for 18 h at 37 ° C either directly in the food samples or after the addition of an enrichment broth (90 ml/sample) and the presence of S. aureus in these samples was investigated by the immunosensor assay and plate counts. The enrichment broths used were BHI broth or a laboratory-formulated liquid version of Baird-Parker agar which contained (g/l: tryptone, 10; Lab-Lemco powder, 5; yeast extract, 1; sodium pyruvate 10; glycerine, 12; lithium chloride, 5; pH 7.0). Chicken ~amples naturally contaminated with S. aureus were also examined by the immunosensor assay and plate counts. Electrochemical immunosensor assay. This is schematically outlined in Fig. 1. Microtitre plate wells were coated with 200 txl human IgG (10~xg/ml) in 0.05 M sodium carbonate buffer (g/l: NazCO3, 1.5; NaHCO3, 2.93; pH 9.6) at room temperature for 1 h. Test antigen dilutions (pure cultures of S. aureus diluted in PBSTM or food samples) were boiled for 15 min to destroy endogenous phosphatase activity and partially extract protein A from the bacteria. After washing plates with PBST, antigen dilutions were added (200 ~xl)to the microtitre plate wells (four replicates). After incubation for 90 min at room temperature, with gentle shaking, 50 pJ rabbit anti-protein A antibody (final concentration 5 lxg/ml in PBSTM) was added and the incubation continued for 1 h. A 1:160 dilution of goat anti-rabbit IgG-alkaline phosphatase conjugate in PBSTM (50 ~1) was added to each well and after 30 min incubation with gentle shaking, plates were washed with PBST and 200 pJ of the substrate solution (2.5 mM phenyl phos-

244 phate in pH 9.6 carbonate buffer) was added. After incubation for 1 h at 37 ° C, the product of the reaction (phenol) was detected electrically using an amperometric platinum anode - Ag/Ag C1 reference electrode system (Rank Bros., Cambridge, Cambs, UK) and a potentiostat (Ministat model, Thomson Electrochem, Newcastle-upon-Tyne, UK) to polarise the platinum electrode at + 870 mV with respect to the reference electrode, to electrochemically oxidise and detect the phenol (Wehmeyer et al. 1985). The reaction chamber contained 1 ml carbonate buffer which was stirred by a magnetic flea and the Ag/AgC1 reference electrode was covered with a 1-cm2 piece of tissue paper soaked in saturated KCI as the electrolyte. On addition of a sample (195 ~tl) from the microtitre plate, the current generated was measured as the voltage drop over a 2200 ~ resistor using a microvolt digital multimeter (model 177, Keithly Instruments, Reading, Berks, UK). The analogue output of the multimeter was plotted on a chart recorder (Model CR600, J. J. Lloyd Instruments, Southampton, Hants, UK). With each sample, the current reached a maximum value 5-15 s after sample addition. Between each determination, the electrode was cleaned with distilled water. Appropriate positive and negative controls were included and the assay took 4-5 h complete.

130 120 1t0 100 90 80 70 60 50 40

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Time (seconds)

Results

Electrochemical detection o f pure cultures o f S. aureus

T h e e l e c t r o c h e m i c a l s t e p in t h e i m m u n o s e n s o r r e l i e d o n d e t e c t i n g p h e n o l , w h i c h is o x i d i s e d at a n o d i c p o t e n tials a b o v e + 7 5 0 m V ( D o y l e et al. 1984; W e h m e y e r et al. 1985). T h e p h e n o l is p r o d u c e d b y a l k a l i n e p h o s p h a tase f r o m p h e n y l p h o s p h a t e , w h i c h is e l e c t r o i n a c t i v e at p o s i t i v e p o t e n t i a l s . T h e o p t i m u m p o t e n t i a l for t h e oxid a t i o n o f p h e n o l in o u r i m m u n o s e n s o r a s s a y w a s d e t e r m i n e d to b e + 870 m V (results n o t s h o w n ) . This a s s a y o f p h e n o l w a s v e r y fast (Fig. 2) a n d t h e q u a n t i t y o f p h e n o l was p r o p o r t i o n a l to t h e cell d e n s i t y o f p r o t e i n A-

Fig. 2. Typical response curves generated by the homogeneous amperometric immunosensor specific for protein A by the addition of three different concentrations of Staphylococcus aureus NCDO 949; A, 2.0 × 105 cfu/ml; B, 1.5 × 10 6 cfu/ml; C, 2.5 × 107 cfu/ml. Each sample (195 ~tl) was injected into the electrochemical buffer chamber at time zero (arrowed) and the current generated (hA) from the oxidation of phenol calculated

b e a r i n g S. aureus. T h e s e m i - h o m o g e n e o u s i m m u n o s e n s o r p r o v e d to b e quite r e l i a b l e a n d gave e s s e n t i a l l y simi l a r r e s p o n s e s with f o u r d i f f e r e n t p r o t e i n A - b e a r i n g S. aureus strains (Fig. 3A, B). T h e a s s a y was q u a n t i t a t i v e b e t w e e n 104 a n d 107 c f u / m l a n d t h e b a c k g r o u n d curr e n t o f ca. 7 n A s u g g e s t e d m i n i m u m n o n - s p e c i f i c a b -

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Fig. 3. A Relationship between the current generated by the homogeneous amperometric immunosensor specific to protein A and the cell densities of pure cultures of different strains of S.

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aureus: O, NCDO 949; O, NCDO 1022; I , NCDO 1499; [3, NCDO 2044. B As A but using an equal number of the four NCDO strains of S. aureus

245 sorption and little non-specific interference from the bacterial cultures. 2.0

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E l e c t r o c h e m i c a l d e t e c t i o n o f S. a u r e u s in f o o d s S. a u r e u s N C D O 949 was successfully detected and

enumerated in samples of milk, cheese, beef and chicken by the semi-homogeneous immunosensor assay (Fig. 4). The sensitivity of the assay was reduced with food samples requiring at least l0 s c f u / g food for reliable detection. With cheese, beef and chicken there was however minimal and consistant background currents (ca. 7 nA) suggesting little non-specific absorption. Milk gave a lower background v a l u e (ca. 4 nA) compared to the other food samples, which were however internally constant. This was perhaps due to its viscosity or that it blocked non-specific absorption to a greater extent than other food samples (c. f. the use of skimmed milk as a c o m m o n blocking agent). Similar resuits were obtained when a mixture of the four strains of S. a u r e u s were inoculated into food samples (results not shown). When samples of milk or beef were inoculated with low cell numbers (ca. 1-2 c f u / g or/ml) of S. a u r e u s N C D O 949, to simulate low levels of contamination of food samples, incubation of these samples or samples homogenised in B H I was sufficient to increase the numbers of organisms to allow successful detection of S. a u r e u s cells by the immunosensors (Table 1). The resuits were quantitative and essentially similar to Fig. 4. The immunosensor was also used to screen directly naturally contaminated chicken samples for protein Abased S. aureus. Just under half proved positive by plate count and the immunosensor assay (Table 1). The reliability of the immunosensor for directly detecting

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Fi~. 4. Relationshipbetween~h¢ current ~enera~¢dby the homo~eneous amperometfic ~mmunosensorspecific to protein A and the cell density of S. ~reus NCDO 949 inoculated into samples of foods: ~, milk; ~, cheese; ~, chicken; O, beef

low numbers of protein A-bearing S. a u r e u s in samples of chicken was investigated further using a set of nonartificially contaminated samples (Table 1). Samples of chicken were incubated for 18 h at 37 ° C directly or preenriched in BHI broth or in the Baird-Parker agarbased broth. This resulted in the positive detection of samples contaminated with S. a u r e u s that appeared to be negative when examined by the plate count method prior to pre-enrichment (Table 1). The good correlation

Table 1. Use of the homogeneous electrochemical immunosensor for detecting protein A-bearing Staphylococcus aureus in food samples Sample type

Sample number

Pre-enrichmenta

Log~0 plate counts b after enrichment

Logao current (hA) after enrichment

Logao cell densityc by immunosensor

A

A1 A2 A3 A4 A5 A6 A7 A8-All A12-A16 B1 B2 B3 B4

Chicken Chicken BHI BHI BPB BPB BPB --BHI BPB BHI Milk

7.36 6.48 3.49 7.20 7.28 < 2.00 < 2.00 --7.60 7.35 7.00 6.55

1.55 1.20 0.90 1.45 1.55 0.85 0.90 1.15-1.25 0.80-0.90 1.75 1.65 1.50 1.05

6.65 5.15 3.90~ 6.45 6.65 < 3.30 < 3.90 5.10-5.30 < 3.20- < 3.90 7.05 6.85 6.55 6.30

B

Type A samples were uninoculated samples of chicken that were initially negative by the plate count (< 102 cfu/g) and the immunosensor assay. Type B samples were spiked with approximately 1-2 cfu/g or ml of S. aureus 949 and pre-enriched: BI and B2 (chicken); B3 (beef); B4 (milk) a Pre-enrichment was performed by incubation at 37° C for 18 h

either in food samples or by addition of Brain Heart Infusion (BHI) or Baird Parker Broth (BPB) to food samples b On Baird Parker agar as described c Calculated from standard curves (e.g. Fig. 4) prepared using an equal number of the four strains of S. aureus. The negative control levels of log10 current were approximately 0.85-0.90

246 between the current generated by the immunosensor and plate counts illustrates the potential usefulness of this simple homogeneous system as a reliable 24-h detection method for food samples containing low numbers of protein A-bearing S. aureus as it can detect ceils which cannot directly be detected by plate counts but which are later shown to be positive by plate counts after the sample has been enriched.

Discussion

Simple and labour-saving rapid methods for detecting pathogenic agents in foods are needed. It is unlikely that any single approach will be a panacea but the assay described in this paper has several attractive features. The assay is based on the concept of the immunosensor (Janata 1975; Aizawa et al. 1980), which has received considerable attention, primarily for medical applications. There are only a few reports of immunosensors for detecting microorganisms (Libby and Wada 1989; Mirhabibollahi et al. 1990a, b). In the past there has been interest in achieving the greatest possible sensitivity in immunoassays (Swaminathan et al. 1985; Libby and Wada 1989). However, in reality no rapid method has been developed that is capable of directly detecting very low numbers of pathogenic organisms in foods that are of concern. Therefore, there are arguments in favour of facilitating the development of simple and automatable rapid methods, rather than always striving for optimal sensitivity. Indeed, unless a rapid method can directly and specifically detect as few as 1-10 cfu/25 g food, some pre-enrichment step, usually of 18 h, is essenti~il to increase the number of target organisms to detectable levels. The method described in this paper is based on an amperometric near-homogeneous ELISA that contained no labour-intensive and time-consuming washing steps during the immunological reactions. The simplicity of this immunosensor is comparable with previously described homogeneous immunoassays (Gibbons et al. 1985; Di Gleria et al. 1986; Anderson 1987; Kjellstr6m and Bachas 1989) and the repeatability of experiments and the reproducibility of results made the assay very reliable (Figs. 2, 3, 4 and Table 1). The use of microtitre plates as solid supports for the assay could also favour automation. The sensitivity of this semi-homogeneous assay (ca. > 105 cfu/g) is less than the sensitivity of the heterogeneous assay (> 103/g) (Mirhabibolahi et al. 1990b) but this is a trade-off against ease of operation. After pre-enrichment, the semi-homogeneous assay is certainly sensitive enough to detect very low numbers of S. aureus cells in foods, which could not be detected by direct plating of samples and incubation of plates for 2 days (Table 1). This could be a very attractive aspect for detection of other pathogens of concern (e.g. S a l m o n e l l a spp.). The electrochemical step was based on the detection of phenol at an anodic potential (+ 870 mV). Phenol is not normally expected to be present in food sampies or microbiological media and this method should,

and appears to, suffer little interference from samples. Previous immunoassays employing this principle (Doyle et al. 1984; Wehmeyer et al. 1985) incorporated column or liquid chromatography steps. In this paper we have been able to successfully apply this method directly to samples and the electrochemical detection step was very simple and rapid (about 2 min/sample). The method uses simple and reusabl6 electrodes with inexpensive and easy-to-operate equipment. It can be envisaged that custom-designed microelectrodes and dataprocessing equipment could easily be developed. This, combined with the use of microtitre-plate-based assays, could ensure a fast output of samples. The detection of protein A-bearing S. aureus was used as a model system in this study. It should be emphasised that protein A-deficient strains (which are few) will not be detected and, although protein A is an important invasive and pathogenic factor (Koenig 1972), there is no strict relationship between the presence of protein A and the pathogenicity of a particular strain of S. aureus. Nevertheless, the semi-homogenous immunosensor could be used as a simple and reliable screen for protein A-bearing S. aureus strains in foods. It is an obvious step to make the system specific for other food-borne pathogens or toxins by the use of the appropriate immunological reagents. It should also be emphasised that a feature of immunological detection methods for foods is that, where heavy contamination with the target organism that had been subsequently killed (e.g. by heat treatment) occurs, the organisms will still be detected, even though they are non-viable. The work described in this paper was sponsored by the Ministry of Agriculture, Fisheries and Foods, Open Contract Number: CSA 1200.

Acknowledgement.

References

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