The effect of Bacillus subtilis and Streptococcus faecalis var. liquefaciens on staphylococcal enterotoxin A activity

International Journal of Food Microbiology, 2 (1985) 211- 218 Elsevier

211

JFM 00063

The effect of Bacillus subtifis and Streptococcus faecafis var. liquefaciens on staphylococcal enterotoxin A activity S.M.

Daoud and J.M. Debevere

*

Faculty of Agricultural Sciences, State University of Ghent, Coupure 653, 9000 Ghent, Belgium

(Received 9 November 1984; accepted 9 March 1985)

The effect of growing Bacillus subtilis and Streptococcus faecalis vat. liquefaciens on staphylococcal growth, thermonuclease (TNase) activity and enterotoxin A (SEA) activity was investigated in liquid media and in foods. Abundant growth of B. subtilis or S. faecalis vat. liquefaciens decreased purified SEA concentrations during a 2-day incubation in brain heart infusion broth (BHI) and supernatant fluid by 89 and 67%, respectively. Both staphylococcal TNase activity and SEA concentrations decreased when S. aureus was cultivated in the presence of B. subtilis or S. faecalis var. liquefaciens. Staphylococcal TNase activity decreased by 75 and 78% in the presence of B. subtilis and S. faecalis var. liquefaciens, respectively, while SEA activity decreased by 95 and 65%, respectively, in the presence of those test strains. The results obtained with artificially contaminated Sterile chicken or beef samples were similar to those obtained with BHI broth. S. aureus grew very well in heated vegetables. TNase was undetectable, although SEA could be detected. In the presence of B. subtilis or S. faecalis var. liquefaciens, SEA activity was decreased to a non-detectable concentration. Key words: Bacillus subtilis; Enterotoxin A; Staphylococcus aureus; Streptococcus faecalis liquefaciens; Thermonuclease

var.

Introduction Staphylococcus aureus is one of the most c o m m o n bacteria causing food p o i s o n ing. The toxic properties of staphylococcal strains correlate well with the p r o d u c t i o n of T N a s e a n d coagulase (Lachica et al., 1969; Simkovicova a n d Gilbert, 1971; Sperber a n d Tatini, 1975). Close correlations b e t w e e n S. aureus growth a n d T N a s e p r o d u c t i o n a n d between T N a s e a n d e n t e r o t o x i n p r o d u c t i o n have been d e m o n s t r a t e d in various food samples (Chesbro a n d A u b o r n , 1967; Lachica et al., 1972). It has also been shown that c o n d i t i o n s affecting e n t e r o t o x i n c o n c e n t r a t i o n in cheese exerted a similar effect o n T N a s e activity. U n d e r all tested conditions, T N a s e was detected whenever S. aureus grew, whether or not e n t e r o t o x i n was f o u n d to be present. In contrast, Lee et al. (1975) indicated that n o nuclease activity could be

* To whom correspondence should be sent 0168-1605/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

212 detected from toxic dough samples contaminated with a S. aureus strain. Daoud and Debevere (1984) have demonstrated that a negative TNase test is not a reliable indicator of the absence of staphylococcal growth and concomitant enterotoxin production in foods containing B. subtilis or S. faecalis var. liquefaciens since these bacteria are able to inactivate TNase. Demonstration of staphylococcal enterotoxin (SE) in suspect food has a high evidential value for the confirmation of an outbreak of staphylococcal food poisoning. Quantities of 0.5 to 5.0 #g of SE ingested with food can cause symptoms such as vomiting and diarrhea (BergdoU, 1970). To date, nanogram quantities of SEA, SEB, and SEC have been successfully measured in food extracts by an enzyme-linked immunosorbent assay (Kuo and Silverman, 1980). The number of S. aureus cells necessary for SE detection varies. Barber and Deibel (1972) reported S. aureus counts of 1 × 107-4 X 107/g as minimal numbers producing detectable amounts of SEA, 8.3 x 10S/g for SEB and 2.3 x 10S/g for SEC. Casman and Bennett (1965) reported that S. aureus counts of 1 x 106-3 x 10 9 were found in foods incriminated in food poisoning. The normal microflora of non-processed foods usually inhibit S. aureus growth and subsequent enterotoxin production. S. aureus is capable of growing and producing toxin specially in heated or cured foods, recontaminated with low numbers of S. aureus. Thermoresistant spores of B. subtilis can survive heat treatment in foods. During storage under unfavorable conditions, sporulation occurs and vegetative cells of B. subtilis can grow together with S. aureus in post-contaminated foods. S. faecalis var. liquefaciens is one of the indicator organisms which can also be present in recontaminated foods. The aim of this investigation was to study the effect of growing B. subtilis and S. faecalis var. liquefaciens, isolated from food, on staphylococcal growth and enterotoxin A activity in broth and foods.

Materials and Methods

Cultures

Cultures of S. faecalis var. liquefaciens and B. subtilis and a coagulase positive TNase and enterotoxin A-producing S. aureus (V17) were kindly provided by the Institute of Hygiene and Epidemiology, Brussels, Belgium. Preparation of inocula

The strains were grown overnight in brain heart infusion broth (BHI) at 37°C. The cells were washed 3 times with 0.85% saline by centrifugation and appropriate suspensions were made based on optical density.

213

Extraction of SE The extraction procedure of Notermans et al. (1983) was used. 100 g of food was homogenized in a Waring blender with an equal amount of distilled water. The mixture was adjusted to pH 4.5 using 1 N HCI. Subsequently, the food slurry was centrifuged (10000 × g at 5°C for 15 rain). The solidified fat was removed and the supernatant liquid (approx. 100 ml) adjusted to pH 7.2 using 1 N NaOH. If precipitation occurred, the extract was centrifuged (10000 × g). Thereafter, the extract was concentrated by dialysis against a solution of polyethylene glycol (PEG, 35~ w/v).

The ELISA test The sandwich ELISA technique was used for determination of SE as described by Biahning-Pfaue et al. (1981). The IgG used for coating and the IgG peroxidase conjugate were kindly provided by Dr. S. Notermans, National Institute of Public Health, Bilthoven, The Netherlands.

Extraction and assay of TNase The TNase extraction procedure of Chesbro and Auborn (1967) was used. TNase activity was quantitatively assayed by the turbidimetric method of Erickson and Deibel (1973) and qualitatively by the agar diffusion test (ADT) of Lachica et al. (1971).

Plating procedure Baird-Parker's medium, plate count agar, and Slanetz and Bartley agar were used for enumeration of S. aureus, B. subtilis and S. faecalis var. liquefaciens, respectively.

Breakdown of SEA by B. subtilis and S. faecalis var. liquefaciens B. subtilis and S. faecalis var. liquefaciens were inoculated separately into BHI broth (initial cell concentration approx. 105 cells/ml) containing 333 ng of purified SEA (Sigma) per ml. After incubation at 37°C for 24 h, SEA was assayed by ELISA. Breakdown of naturally produced SEA and TNase by B. subtilis and S. faecalis oar liquefaciens S. aureus (V17) was inoculated into 100 ml broth in 500 ml Erlenmeyer flasks and incubated at 37°C for 24 h. Supernatant fluid was membrane filtered (Gelman GN-6, 0.45 #m). This supernatant was inoculated with B. subtilis and S. faecalis vat. liquefaciens separately (initial cell conc. approx. 105 cells/ml). After a 24 h incubation at 37°C, cell counts of the test strains and the SEA concentration were determined.

214

Influence of B. subtilis and S. faecalis oar. liquefaciens on SEA activity in B H I broth Enterotoxin A production by S. aureus (V17) in the.presence of B. subtilis or S. faecalis var. liquefaciens (initial cell conc. approx. 104 cells/ml) was studied in BHI broth at p H 7.0. The inoculated Erlenmeyer flasks were kept at 37°C under aerobic conditions. After 24 h, both the final p H and the number of viable cells of S. aureus and other test strains, TNase activity, and enterotoxin A concentration were estimated.

Studies in food samples Enterotoxin A production by S. aureus (V17) in the presence of B. subtilis or S. faecalis var. liquefaciens was studied in sterilized (120°C for 15 min) minced meat, chicken and vegetables in the same way as in BHI broth.

Results and Discussion Table I shows the results of the breakdown of SEA in BHI in the presence of B. subtilis and S. faecalis var. liquefaciens. The data indicate that abundant growth of B. subtilis or S. faecalis vat. iiquefaciens decreased the SEA concentrations during incubation for 2 days in BHI containing 333 ng per ml of enterotoxin. SE concentrations decreased by 89 and 67% in the presence of B. subtilis and S. faecalis var. liquefaciens, respectively. The breakdown of purified SEA by S. faecalis var. liquefaciens was nearly the same as that found by Chordash and Potter (1976). Furthermore, our results show that B. subtilis also caused a significant breakdown of purified SEA in contrast to the results of Chordash and Potter (1976) who found that proteolytic bacteria had no effect on recoverable toxin levels. Data on the breakdown of naturally produced SEA by B. subtilis and S. faecalis var. liquefaciens are given in Table II. In addition, TNase activity has also been recorded. Abundant growth of B. subtilis or S. faecalis var. liquefaciens decreased both TNase and SE concentrations during incubation for 2 days in supernatant fluids. SEA concentrations decreased by 91 and 67% in the presence of B. subtilis or S. faecalis var. liquefaciens, respectively. On the other hand, B. subtilis and S.

TABLE 1 Breakdown of SEA in BHI containing 333 ng SEA/ml by B. subtilis and S. faecalis var. liquefaciens Test strains BHI + SEA + SEA + B. subtilis + SEA + S. faecalis var. liquefaciens

ng of SEA/ml 333 37 111

215 TABLE II Breakdown of naturally produced SEA and TNas¢ by B. subtilis and S. faecalis vat. liquefaciens at 37°C for 24 h Test strains

Final log~0 number of the test strains/ml

TNase units/ rnl "

Amount of SEA (ng/ml)

Supernatant fluid Supematant fluid + B. subtilis Supematant fluid + S. faecalis var. liquefaciens

9.2

48 12

108 10

8.8

10

36

a TNase units/ml =/~g of substrate depolymerized per ml x dilution factor/time of incubation (min).

faecalis var. fiquefaciens caused n e a r l y the s a m e degree o f i n a c t i v a t i o n o f s t a p h y l o coocal T N a s e activity. T h e s e findings are in c o m p l e t e a g r e e m e n t with those o b t a i n e d in earlier e x p e r i m e n t s ( D a o u d a n d D e b e v e r e 1984). D a t a on s t a p h y l o c o c c a l g r o w t h a n d e n t e r o t o x i n A activity in B H I , artificially c o n t a m i n a t e d with B. subtilis or S. faecalis var. liquefaciens, are s u m m a r i z e d in T a b l e III. W h e n B. subtilis o r S. faecalis var. liquefaciens were g r o w n in B H I with S. aureus, the s t a p h y l o c o c c i increased to a p p r o x i m a t e l y 108 c e l l s / m l , whereas the c o n c e n t r a t i o n o f S E A decreased b y 95% or 65%, respectively. T N a s e activity was also significantly d e c r e a s e d in the presence o f B. subtilis a n d S. faecalis var. liquefaciens. D a t a illustrating s t a p h y l o c o c c a l g r o w t h a n d e n t e r o t o x i n A c o n c e n t r a t i o n in heated, m i n c e d f o o d samples, artificially c o n t a m i n a t e d with B. subtilis a n d S. faecalis vat. liquefaciens are s u m m a r i z e d in T a b l e IV. T h e s e d a t a clearly show that S. aureus grows well in h e a t e d m i n c e d food s a m p l e s in the presence of B. subtilis o r

TABLE

IIl

Staphylococcal growth and enterotoxin A activity in BHI artificiallycontaminated with B. subtilis and S. faecali$ vat. liquefaciens Test strains

Final pH

Final log10 number of S. aureus/ml

Final log10 number of test s t r a i n s / m i

8.9 8.5 8.7

8.3 8.1

TNase (units/ml)"

Amount of SEA (ng/ml)

70 17 15

144 7 50

BHI

+ S. aureus + $. aureus + B. subtilis + S. aureus + S. faecalis var. liquefaciens

6.60 7.00 6.95

" TNase units/ml ~/tg of substrate depolymerized per mi × dilution factor/time of incubation (rain).

216 TABL E IV Staphylococcal growth and enterotoxin A concentration in heated minced food samples artificially contaminated with the test strains Inoculated a

Incubation at 37°C for 48 h Final pH

Final log10 number of S. aureus/g

Final loglo number of the test strains/g

TNase units/ml b

A mount of SEA /~g/100 g

Minced beef 1 2 3

7.10 7.00 6.90

9.9 9.4 9.5

9.7 9.3

40 9 21

38 5 22

Minced chicken 1 2 3

6.95 6.90 6.85

9.2 9.3 9.0

9.5 9.1

45 10 15

35 4 15

Minced peas 1 2 3

6.50 6.70 6.30

9.0 9.3 8.9

9.0 9.2

ND ND ND

3 ND ¢ ND

Minced string beans 1 2 3

6.30 6.50 6.20

8.5 8.7 8.5

8.9 9.0

ND ND ND

3.2 ND ND

Minced lettuce 1 2 3

6.60 6.90 6.50

8.3 8.6 8.5

9.0 9.2

ND ND ND

ND ND ND

a 1 = S. aureus, 2 = S. aureus + B. subtilis, 3 ~ S. aureus + S. f a e c a l i s var. liquefaciens.

b TNase u n i t s / m l = ~g of substrate depolymerized per ml × d i l u t i o n f a c t o r / t i m e of incubation (min). c N D = not detectable.

S. faecalis var. liquefaciens. However, SEA and TNase activity were decreased in the presence of B. subtilis or S. faecalis var. liquefaciens. These results were similar to those obtained in the experiments with BHI (Table III). Although the growth of S. aureus is vegetables is excellent, the SEA concentration is, however, sharply decreased. SEA could be detected in beef and chicken meat samples artificially contaminated with B. subtilis or S. faecalis var. liquefaciens. However, in vegetables artificially contaminated with B. subtilis or S. faecalis var. liquefaciens, SEA could not be detected. Hence, it can be concluded that B. subtilis or S. faecalis var. liquefaciens had an effect on SEA concentration in BHI and heated minced food samples. Reduced levels of enterotoxin may be the result of competition for an essential nutrient for enterotoxin formation or a breakdown of the enterotoxin by these test strains. It is

217 also possible that an" inhibiting substance was p r o d u c e d in quantities sufficient for the inhibition of enterotoxin formation, without significant reduction of staphylococcal growth. A n important conclusion from these investigations is that enterotoxin A and T N a s e production by S. aureus are seen to be dependent on the food itself. It is clearly demonstrated (Table IV) that despite the good growth of S. aureus, toxin concentration is sharply decreased while T N a s e activity is not detectable by means of available methods for T N a s e assessment. Furthermore, it can be concluded that despite the decrease in SEA activity in heated minced beef and chicken in the presence of B. subtilis or S. faecalis vat. liquefaciens, these food samples still contain sufficient quantities of SEA to cause food poisoning. In contrast, SEA could not be detected in heated minced vegetable artificially contaminated with B. subtilis or S. faecalis vat. fiquefaciens. Based u p o n this investigation and previously reported results ( D a o u d and Debevere, 1984) we conclude that the safety of certain food products cannot exactly be predicted. Therefore, suspected foods have to be tested for their ability to support enterotoxin formation by S. aureus.

Acknowledgements The authors wish to express their appreciation to Dr. S. N o t e r m a n s ( L a b o r a t o r y of Zoonoses and F o o d Microbiology, National Institute of Public Health, Bilthoven, The Netherlands) for providing the reagents of E L I S A and to Mr. F. Shapiro for editorial assistance.

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