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Effect of growth of a commercial starter culture on growth of Staphylococcus aureus and thermonuclease and enterotoxins (C1 and C2) production in broth cultures
International Journal ofFoodMicrobiology, 6 (1988) 107-114 Elsevier
107
JFM00190
Effect of growth of a commercial starter culture on growth of Staphylococcus aureus and thermonuclease and enterotoxins (C1 and C2) production in broth cultures A. Otero, M.C. Garcia, M.L. Garcla and B. Moreno Veterinary Faculty, University of Lern, Lebn, Spain
(Received 14 April 1987; revised version received 16 September 1987; accepted 13 November 1987)
Staphylococcus aureus strains FRI 137 (enterotoxin C l producer) and FRI 361 and L 2 (enterotoxin C 2 producers) were grown alone and in the presence of a mixed commercial starter culture (Streptococcus lactis, Streptococcus cremoris and Streptococcus lactis subsp, diacetylactis). Lactic acid bacteria had a slight inhibitory effect on S. aureus population and only during the late stages of growth. In contrast, enterotoxin synthesis was strongly inhibited, inhibition at 18 h being 89% (FRI 137), 80% (FRI 361) and 69% (L 2). Enterotoxin C 1 was produced and accumulated during all phases of growth in pure and mixed culture, but associative growth resulted in reduction of enterotoxin C 2 after 24-36 h. In mixed culture, high producers of thermonuclease (FRI 137 and L 2) showed an early decrease in enzyme activity followed by an increase, but it never reached levels attained in pure culture. Thermonuclease was detected whenever enterotoxin was detected, but production curves did not parallel each other.
Key words: Staphylococcus aureus; Starter cultures; Enterotoxins C~ and C2; Thermonuclease
Introduction I n v e s t i g a t i o n s o f m u t u a l i n t e r a c t i o n s in m i x e d m i c r o b i a l p o p u l a t i o n s m a y h e l p to u n d e r s t a n d t h e b e h a v i o r of c e r t a i n b a c t e r i a l species in f o o d s . T h e c o m p o s i t i o n o f the m i c r o b i a l f l o r a o f f o o d s is n o r m a l l y an i m p o r t a n t f a c t o r i n f l u e n c i n g s t a p h y l o c o c c a l g r o w t h a n d p r o d u c t i o n o f t h e r m o n u c l e a s e a n d e n t e r o t o x i n s ( S m i t h et al., 1983). It also a p p e a r s t h a t f o o d m i c r o o r g a n i s m s c a n a f f e c t the s u r v i v a l of S t a p h y l o coccus a u r e u s cells a n d s t a b i l i t y of p r e f o r m e d e n t e r o t o x i n s ( C h o r d a s h a n d P o t t e r , 1976; D a o u d a n d D e b e v e r e , 1985). T h e r e is c o n s i d e r a b l e e v i d e n c e t h a t m a n y l a c t i c a c i d b a c t e r i a a r e a n t a g o n i s t i c t o w a r d S. a u r e u s d u r i n g a s s o c i a t i v e g r o w t h ( G i l l i l a n d a n d S p e c k , 1974). T h e i n t e n s i t y o f the a n t a g o n i s m is d e t e r m i n e d b y the c o m p o s i t i o n o f the s t a r t e r c u l t u r e ,
Correspondence address: B. Moreno, Department of Food Hygiene and Food Microbiology, Veterinary Faculty, University of Le6n, 24007-Le6n, Spain.
0168-1605/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
108 the strain of S. aureus tested, the proportion of bacteria inoculated and the environmental conditions (medium, temperature, etc.). The way in which this antagonism operates may include one or more of the following mechanisms: (1) physicochemical changes in the medium (pH alteration, formation of reducing compounds, etc.), (2) nutritional competition, and (3) formation of antimicrobial products of bacterial origin (organic acids, hydrogen peroxide, antibiotics, etc.). The predominant mechanism depends on conditions of testing and time, organisms used as effectors and the strain of S. aureus. The purpose of this work was to determine the effect of growth of a commercial starter on staphylococcal growth and production of thermonuclease and enterotoxins C 1 and C 2 in a liquid medium.
Materials and Methods
Staphylococcus aureus cultures
Strains FRI 137 (enterotoxin C 1 producer) and FRI 361 (enterotoxin C 2 producer) were obtained from Professor M.S. Bergdoll of the Food Research Institute, University of Wisconsin-Madison, U.S.A. Strain L 2 (C 2 producer) was isolated from ovine mastitic milk (Gutirrrez et al., 1982). Isolates were subcultured three times (37 o C; 24 h) in brain heart infusion broth (BHI, Oxoid) immediately before use. The inoculum was prepared by washing twice with sterile 0.1% peptone water ( w / v ) and centrifugation at 12200 × g for 10 min at 5 ° C using a Sorvall RC-5B refrigerated centrifuge (Du Pont Company, U.S.A.). Starter culture
The mixed-strain starter used (HM1) was provided by Miles-Martin, S.A.E., Madrid, Spain. It contained Streptococcus lactis, Streptococcus cremoris and Streptococcus lactis subsp, diacetylactis. The inoculum was prepared by subculturing in sterile 12% skim milk (Oxoid) and incubation at 2 3 ° C for 16 h. The activity of the starter culture was evaluated on the basis of acidity developed in skim milk (Oxoid), taking into account the supplier's recommendations. Associative growth
Each S. aureus strain was inoculated (approx. 105 colony-forming units (cfu)/ml, final concentration) into two 500 ml-Erlenmeyer flasks containing 200 ml APT broth (Difco). One of the flasks was additionally inoculated with about 105 c f u / m l of the starter culture. The control and associative cultures were incubated (without shaking) for 48 h at 30 o C. Samples were withdrawn from both flasks at various intervals (0, 8, 12, 18, 24, 36 and 48 h) and determination of cfu/ml, enterotoxin and thermonuclease, and pH measurements were made. Samples for thermonuclease and enterotoxin detection (10 ml) were centrifugated for 10 min at 12200 × g at
109 5 ° C and frozen at - 3 0 ° C for later analysis. Percentages of inhibition were calculated using the formula described by Ibrahim (1978). Each experiment was performed twice and each sample assayed in duplicate. Data reported correspond to mean values _+ standard deviations. Colony-forming units
The c f u / m l of S. aureus was obtained by plating 0.1 ml of the appropriate dilutions on Baird-Parker medium (Oxoid). The plates were incubated at 37 ° C for 48 h (ICMSF, 1978). Lactic acid bacteria were enumerated on MRS agar (Oxoid) adjusted to pH 5.5 (Kitchell and Shaw, 1975), and incubated at 32°C for 3 days. Thermonuclease assay
Thermonuclease activity was determined by the quantitative procedure of Ibrahim (1981). Before testing, each supernatant was heated in a boiling bath for 15 min and its pH adjusted to 10. A standard curve was prepared using Micrococcal Nuclease (Sigma, 100/zmolar units/mg) and Salmon Sperm DNA (Difco). Detection of enterotoxins
Enterotoxins were detected using the single diffusion tube method as described by Kato et al. (1966), the double gel diffusion slide method proposed by Casman et al. (1969) and the sandwich ELISA technique of Fey et al. (1984). The reagents for ELISA test were obtained from W. Bommelli, Bern, Switzerland. Samples giving negative results with the microslide technique were concentrated by dialyzing against 30% (w/v) Carbowax 20M (Serva, Heildelberg, F.R.G.) and retested.
Results
The growth, enterotoxin production and thermonuclease activity of S. aureus strains FRI 137, FRI 361 and L 2 grown alone and in association with the starter culture are shown in Tables I, II and III, respectively. The presence of lactic acid bacteria had an inhibitory effect on the population of S. aureus but only after 18 h or more of incubation. The effect varied with the strain; after 48 h incubation, the percentages of inhibition were 18% (FRI 137), 11% (FRI 361) and 7% (L 2). Enterotoxin synthesis was strongly inhibited by the starter culture. When strain FRI 137 was grown in mixed culture, the level of enterotoxin C 1 at 18 h was 11% of the amount produced when the culture was grown alone; the corresponding figures for strains FRI 361 and L 2 (C 2 producers) were 20 and 31%, respectively. Furthermore, while enterotoxin C 1 was produced and accumulated during all phases of growth (pure and mixed culture), associative growth resulted in an apparent reduction of enterotoxin C 2 after 24-36 h incubation.
5.11 _+0.07 9.46_+0.03 9.49_+0.06 9.59_+0.05 9.28_+0.07 8.65-+0.05 7.78_+0.05
ND 0.13_+0.03 0.17_+0.04 0.25_+0.02 0.37+_0.08 0.39_+0.03 0.46_+0.05
Enterotoxin C 1 (p~g/ml) ND 2.36_+0.17 2.37+_0.19 2.36_+0.15 2.37_+0.15 0.16_+0.04 0.27+0.05
Thermonuclease (~g/ml)
6.96_+0.02 6.40_+0.03 6.00_+0.04 4.78_+0.03 4.81_+0.04 4.77_+0.03 4.81_+0.02
pH
4.81 +0.04 9.23_+0.05 9.70_+0.07 9.51_+0.02 9.57-+0.04 9.41_+0.07 8.70_+0.06
ND a 0.17_+0.03 2.0 _+0.1 2.6 +_0.2 2.9 _+0.4 6.3 +_0.3 10.5 _+0.6
4.78_+0.06 9.15_+0.03 9.68_+0.08 9.59_+0.03 9.46_+0.07 9.81_+0.04 9.78_+0.03
6.96_+0.04 6.49_+0.05 5.77_+0.03 4.79_+0.04 4.95_+0.06 4.67_+0.03 4.65+_0.04
logl0 S. aureus/ml
ND 0.006_+0.002 0.02 +0.005 0.03 - + 0 . 0 1 0.03 _+0.01 0.03 _+0.01 0.03 _+0.01
Mixed culture pH
lOglo S. aureus/ml
Thermonuclease (~g/ml)
Enterotoxin C 2 (/tg/ml)
Pure culture
ND, not detected.
0 8 12 18 24 36 48
Hour
ND 0.05_+0.02 0.30_+0.03 0.52_+0.09 0.50_+0.05 0.32_+0.07 0.15_+0.06
Enterotoxin C 2 (~g/ml)
ND 0.03 _+0 . 0 1 0.01 _+0.005 0.001 _+0.000 0.003_+0.001 0.02 _+0.007 0.05 _+0.01
Thermonuclease (/tg/ml)
6.92_+0.02 6.38_+0.03 5.70_+0.04 4.70_+0.02 4.84_+0.03 4.81_+0.05 4.86_+0.03
pH
Growth and synthesis of thermonuclease and enterotoxin C 2 by Staphylococc~" aureus FR1 361 in pure culture and in mixed culture with the starter HM1 (mean values-+ standard deviations)
TABLE II
6.96_+0.03 6.45_+0.04 6.01 _+0.02 5.12_+0.03 4.91_+0.04 4.68_+0.02 4.69_+0.03
ND " 0.32_+0.03 0.44_+0.04 2.2 _ + 0 . 2 11.8 _ + 0 . 3 14.3 _ + 0 . 4 23.1 _ + 0 . 3
5.00_+0.06 9.45_+0.02 9.75+-0.09 9.57+-0.06 9.67_+0.02 9.81_+0.05 9.53_+0.03
ND 2.37_+0.16 2.38_+0.15 2.36_+0.13 3.12+-0.20 7.05+_0.31 7.04_+0.23
Mixed culture pH
lOgl0 S. aureus/ml
Thermonuclease (/~g/ml)
Enterotoxin C 1 (/zg/ml)
Pure culture
lOglo S. aureus/ml
a ND, not detected.
0 8 12 18 24 36 48
Hour
Growth and synthesis of thermonuclease and enterotoxin C 1 by Staphylococcus aureus FRI 137 in pure culture and in mixed culture with the starter HM1 (mean values_+ standard deviations)
TABLE I
4.79+0.08 9.40+0.03 9.85-+0.07 9.70+0.06 9.63+0.04 9.99_+0.05 9.67+0.02
ND a 0.30_+0.04 0.44-+0.06 1.9 _+0.2 4.0 - + 0 . 3 4.4 _ + 0 . 2 7.2 +0.4
ND 0.21_+0.03 0.82-+0.08 0.81 _+0.07 0.82-+0.06 0.80_+0.08 0.81-+0.05
6.98+0.03 6.49_+0.06 5.70-+0.05 5.07_+0.05 4.85_+0.03 4.68_+0.04 4.75_+0.02
pH
4.88+0.04 9.48_+0.05 9.86_+0.06 9.56_+0.03 9.53_+0.07 9.53_+0.06 9.00_+0.03
log]0 S. aureus/ml
Thermonuclease (/Lg/ml)
log]o S. aureus/ml
Enterotoxin C 2 (~g/ml)
Mixed culture
Pure culture
a ND, not detected.
0 8 12 18 24 36 48
Hour
ND 0.11_+0.08 0.33_+0.06 0.58_+0.09 0.61_+0.06 0.76_+0.02 0.39_+0.08
Enterotoxin C 2 (~g/ml)
ND 0.09+0.01 0.80+0.06 0.81 +0.06 0.28_+0.03 0.27_+0.05 0.47+0.04
Thermonuclease (/t g/ml)
6.96+0.02 6.41+0.04 5.63-+0.03 4.97-+0.05 5.01_+0.02 5.00-t-0.03 5.05-+0.04
pH
Growth and synthesis of thermonuclease and enterotoxin C2 by Staphylococcus aureus L 2 in pure culture and in mixed culture with the starter HM1 (mean values _+standard deviations)
TABLE III
112
The effect of lactic acid bacteria on thermonuclease activity varied with the amount of enzyme produced in pure culture. With strains FRI 137 and L 2 (high producers of thermonuclease), a decrease between 18 and 36 h was observed, followed by an increase between 36 and 48 h. After 18 h incubation, the level of enzyme was always lower than that in pure culture. In contrast, at 8 and 48 h, thermonuclease activity of strain F R I 361 (low producer) appeared to be higher in associative culture than in pure culture. Inhibition of thermonuclease activity was observed between 12 and 36 h. Although the p H of mixed cultures decreased slightly faster, final values were lower in the pure cultures.
Discussion
The starter culture examined showed little inhibitory effect on growth of the three strains of S. aureus included which all attained population levels above ] 0 9 c f u / m ] in mixed cultures. Haines and H a r m o n (1973) found a stronger inhibitory influence of Streptococcus cremoris and Streptococcus lactis on growth of S. aureus strain F D A 243, which produces enterotoxin A. It should be noted that the slight inhibition observed in the present study seemed to occur in the stationary phase i.e. the exponential growth rate was not affected. This observation was also made by Haines and H a r m o n (1973). The effect of starter cultures on growth of S. aureus has been attributed, among other factors, to reduction of p H (Babel, 1977). However, the present results show that inhibition occurred in the late stages of incubation, when p H values of the mixed cultures were equal to or higher than those of pure cultures. Although substantial reduction in enterotoxins C 1 and C 2 were observed the starter culture used failed to prevent enterotoxin production. The ability of lactic bacteria and other organisms to prevent production of staphylococcal enterotoxins A and B in foods and broth has been reported by several authors (Haines and Harmon, 1973; Niskanen and Nurmi, 1976). The effect obtained apparently depends on the type and level of starter culture used as well as the sensitivity of the methods for enterotoxin detection. The inhibiton of production of enterotoxins C1 and C 2 and the decrease in enterotoxin C2 activity during the late stages of incubation in mixed culture may help to explain the low incidence of staphylococcal food-poisoning for Spanish cheeses prepared from sheep milk. In a previous work, 80% of S. aureus strains isolated from ovine mastitic milk in Spain produced enterotoxin C (Guti6rrez et al., 1982). Decrease in enterotoxin activity and even disappearance of toxins in the presence of lactic acid bacteria and other organisms have also been observed by Donnelly et al. (1968) in milk (enterotoxin A), Chordash and Potter (1976) in broth (enterotoxin A), Niskanen and Nurmi (1976) in dry sausage (enterotoxin C~) and Daoud and Debevere (1985) in broth and foods (enterotoxin A). The exact mechanism of the inhibitory effect on enterotoxin formation and the disappearance of toxins in mixed cultures is unknown. D a o u d and Debevere (1985)
113 suggested that it might be due to competition for essential nutrients, production of an inhibiting substance a n d / o r breakdown of the enterotoxin, and Chordash and Potter (1976) indicated that the decrease in enterotoxin A could be related to enzymes or other metabolites acting alone or assisted by a high acidity. One might conclude from the present results that, if lactic acid bacteria can inhibit the growth of pathogens in foods and decrease enterotoxin production, this is an additional desirable parameter when selecting and improving strains of microorganisms to be used for the fermentation of foods. In mixed culture, early decrease in thermonuclease activity could be due to proteolytic enzymes produced by lactic acid bacteria. Inactivation of thermonuclease by Streptococcus faecalis var. liquaefaciens was described by Lachica et al. (1972). Furthermore, Medwid and G r a n t (1980) reported that the above species produces a proteolytic substance called thermonuclease inactivating factor (TIF). Inactivation by Bacillus subtilis has also been reported (Daoud and Debevere, 1985). Our results show that the increase in thermonuclease activity during the late stages of incubation occurred when the colony-forming units of S. aureus had begun to decline. Since thermonuclease production was not observed when the starter culture was grown (without S. aureus) in APT broth at 3 0 ° C for 48 h (data not reported), it is unlikely that the increase could have been caused by the lactic acid bacteria. In associative culture, thermonuclease was detected whenever enterotoxin was detected ( > 1 n g / m l ) ; but the production of these metabolites did not parallel each other. This lack of correlation between the levels of enterotoxin and thermonuclease is consequence of the differences between strains of S. aureus in their ability to produce thermonuclease and enterotoxin. Also, the influence of lactic starter cultures varies with the strain used. Our results suggest that the thermonuclease test is a reliable indicator of S. aureus growth and enterotoxin production in associative cultures. However, this test cannot be used to predict either the number of S. aureus or the amount of enterotoxin accumulated.
Acknowledgements This work was supported in part by a grant (Project No. 4466-79) from the Spanish CAICYT, as well as by funds from Junta de Castilla-Lern (Consejeria de Educacirn y Cultura) and by D i p u t a c i r n Provincial de Lern. We are also indebted to Professor M.S. Bergdoll and to Miles-Martin, S.A.E. for providing enterotoxins and antisera, and the starter culture HM1, respectively.
References Babel, F.J. (1977) Antibiosis by lactic culture bacteria. J. Dairy Sci. 60, 815-821. Casman, E.P., Bennett, R.W., Dorsey, A.E. and Stone, J.E. (1969) The microslide gel double diffusion test for the detection and assay of staphylococcal enterotoxins. Health Lab. Sci. 6, 185-196. Chordash, R.A. and Potter, N.N. (1976) Stability of staphylococcal enterotoxin A to selected conditions encountered in foods. J. Food Sci. 41,906-909.
114 Daoud, S.M. and Debevere, J.M. (1985) The effect of Bacillus subtilis and Streptococcus faecalis var. liquaefaciens on staphylococcal enterotoxin A activity. Int. J. Food Microbiol. 2, 211-218. Donnelly, C.B., Leslie, J.E. and Black, L.A. (1968) Production of enterotoxin A in milk. Appl. Microbiol. 16, 917-924. Fey, H., Pfister, H. and Riiegg, O. 1984. Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C and D. J. Clin. Microbiol. 19, 34-38. Gilliland, S.E. and Speck, M.L. (1974) Antagonism of lactic streptococci toward Staphylococcus aureus in associative milk cultures. Appl. Microbiol. 28, 1090-1093. Guti&rez, L,M., Menes, I., Garcia, M.L., Moreno, B. and Bergdoll, M.S. (1982) Characterization and enterotoxigenicity of staphylococci isolated from ovine mastitic milk in Spain. J. Food Protect. 45, 1282-1289. Haines, W.C. and Harmon, L.G. (1973) Effect of selected lactic acid bacteria on growth of Staphylococcus aureus and production of enterotoxin. Appl. Microbiol. 25, 436-441. Ibrahim, G.F. (1978) Inhibition of Staphylococcus aureus under simulated Cheddar cheese making conditions. Austral. J. Dairy Technol. 33, 102-108. Ibrahim, G.F. (1981) A simple sensitive method for determining staphylococcal thermonuclease in cheese. J. Appl. Bacteriol. 51, 307-312. ICMSF (1978) Microorganisms in foods. 1. Their significance and methods of enumeration. University of Toronto Press, Toronto, 434 pp. Kato, E,, Khan, M., Kujovich, L. and Bergdoll, M.S. (1966) Production of enterotoxin A. Appl. Microbiol. 14, 966-972. Kitchell, A.K. and Shaw, B.G. (1975) Lactic acid bacteria in fresh and cured meats. In: J.G. Cart, C.V. Cutting and G.C. Whiting (Eds.), Lactic Acid Bacteria in Beverages and Foods, Academic Press, London, pp. 209-220. Lachica, R.V.F., Hoeprich, P.D. and Riemann, H.P. (1972) Tolerance of staphylococcal thermonuclease to stress. Appl. Microbiol. 23, 994-997. Medwid, R.D. and Grant, D.W. (1980) Inactivation of staphylococcal thermonuclease by an enzyme-linked factor produced by Streptococcus" faecalis var. liquaefaciens. J. Food Protect. 43, 201-202, 204. Niskanen, A. and Nurmi, E. (1976) Effect of starter culture on staphylococcal enterotoxin and thermonuclease production in dry sausage. Appl. Environ. Microbiol. 31, 11-20. Smith, J.L., Buchanan, R.L. and Palumbo, S.A. (1983) Effect of food environments on staphylococcal enterotoxins synthesis: A review. J. Food Protect. 46, 545-555.
107
JFM00190
Effect of growth of a commercial starter culture on growth of Staphylococcus aureus and thermonuclease and enterotoxins (C1 and C2) production in broth cultures A. Otero, M.C. Garcia, M.L. Garcla and B. Moreno Veterinary Faculty, University of Lern, Lebn, Spain
(Received 14 April 1987; revised version received 16 September 1987; accepted 13 November 1987)
Staphylococcus aureus strains FRI 137 (enterotoxin C l producer) and FRI 361 and L 2 (enterotoxin C 2 producers) were grown alone and in the presence of a mixed commercial starter culture (Streptococcus lactis, Streptococcus cremoris and Streptococcus lactis subsp, diacetylactis). Lactic acid bacteria had a slight inhibitory effect on S. aureus population and only during the late stages of growth. In contrast, enterotoxin synthesis was strongly inhibited, inhibition at 18 h being 89% (FRI 137), 80% (FRI 361) and 69% (L 2). Enterotoxin C 1 was produced and accumulated during all phases of growth in pure and mixed culture, but associative growth resulted in reduction of enterotoxin C 2 after 24-36 h. In mixed culture, high producers of thermonuclease (FRI 137 and L 2) showed an early decrease in enzyme activity followed by an increase, but it never reached levels attained in pure culture. Thermonuclease was detected whenever enterotoxin was detected, but production curves did not parallel each other.
Key words: Staphylococcus aureus; Starter cultures; Enterotoxins C~ and C2; Thermonuclease
Introduction I n v e s t i g a t i o n s o f m u t u a l i n t e r a c t i o n s in m i x e d m i c r o b i a l p o p u l a t i o n s m a y h e l p to u n d e r s t a n d t h e b e h a v i o r of c e r t a i n b a c t e r i a l species in f o o d s . T h e c o m p o s i t i o n o f the m i c r o b i a l f l o r a o f f o o d s is n o r m a l l y an i m p o r t a n t f a c t o r i n f l u e n c i n g s t a p h y l o c o c c a l g r o w t h a n d p r o d u c t i o n o f t h e r m o n u c l e a s e a n d e n t e r o t o x i n s ( S m i t h et al., 1983). It also a p p e a r s t h a t f o o d m i c r o o r g a n i s m s c a n a f f e c t the s u r v i v a l of S t a p h y l o coccus a u r e u s cells a n d s t a b i l i t y of p r e f o r m e d e n t e r o t o x i n s ( C h o r d a s h a n d P o t t e r , 1976; D a o u d a n d D e b e v e r e , 1985). T h e r e is c o n s i d e r a b l e e v i d e n c e t h a t m a n y l a c t i c a c i d b a c t e r i a a r e a n t a g o n i s t i c t o w a r d S. a u r e u s d u r i n g a s s o c i a t i v e g r o w t h ( G i l l i l a n d a n d S p e c k , 1974). T h e i n t e n s i t y o f the a n t a g o n i s m is d e t e r m i n e d b y the c o m p o s i t i o n o f the s t a r t e r c u l t u r e ,
Correspondence address: B. Moreno, Department of Food Hygiene and Food Microbiology, Veterinary Faculty, University of Le6n, 24007-Le6n, Spain.
0168-1605/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
108 the strain of S. aureus tested, the proportion of bacteria inoculated and the environmental conditions (medium, temperature, etc.). The way in which this antagonism operates may include one or more of the following mechanisms: (1) physicochemical changes in the medium (pH alteration, formation of reducing compounds, etc.), (2) nutritional competition, and (3) formation of antimicrobial products of bacterial origin (organic acids, hydrogen peroxide, antibiotics, etc.). The predominant mechanism depends on conditions of testing and time, organisms used as effectors and the strain of S. aureus. The purpose of this work was to determine the effect of growth of a commercial starter on staphylococcal growth and production of thermonuclease and enterotoxins C 1 and C 2 in a liquid medium.
Materials and Methods
Staphylococcus aureus cultures
Strains FRI 137 (enterotoxin C 1 producer) and FRI 361 (enterotoxin C 2 producer) were obtained from Professor M.S. Bergdoll of the Food Research Institute, University of Wisconsin-Madison, U.S.A. Strain L 2 (C 2 producer) was isolated from ovine mastitic milk (Gutirrrez et al., 1982). Isolates were subcultured three times (37 o C; 24 h) in brain heart infusion broth (BHI, Oxoid) immediately before use. The inoculum was prepared by washing twice with sterile 0.1% peptone water ( w / v ) and centrifugation at 12200 × g for 10 min at 5 ° C using a Sorvall RC-5B refrigerated centrifuge (Du Pont Company, U.S.A.). Starter culture
The mixed-strain starter used (HM1) was provided by Miles-Martin, S.A.E., Madrid, Spain. It contained Streptococcus lactis, Streptococcus cremoris and Streptococcus lactis subsp, diacetylactis. The inoculum was prepared by subculturing in sterile 12% skim milk (Oxoid) and incubation at 2 3 ° C for 16 h. The activity of the starter culture was evaluated on the basis of acidity developed in skim milk (Oxoid), taking into account the supplier's recommendations. Associative growth
Each S. aureus strain was inoculated (approx. 105 colony-forming units (cfu)/ml, final concentration) into two 500 ml-Erlenmeyer flasks containing 200 ml APT broth (Difco). One of the flasks was additionally inoculated with about 105 c f u / m l of the starter culture. The control and associative cultures were incubated (without shaking) for 48 h at 30 o C. Samples were withdrawn from both flasks at various intervals (0, 8, 12, 18, 24, 36 and 48 h) and determination of cfu/ml, enterotoxin and thermonuclease, and pH measurements were made. Samples for thermonuclease and enterotoxin detection (10 ml) were centrifugated for 10 min at 12200 × g at
109 5 ° C and frozen at - 3 0 ° C for later analysis. Percentages of inhibition were calculated using the formula described by Ibrahim (1978). Each experiment was performed twice and each sample assayed in duplicate. Data reported correspond to mean values _+ standard deviations. Colony-forming units
The c f u / m l of S. aureus was obtained by plating 0.1 ml of the appropriate dilutions on Baird-Parker medium (Oxoid). The plates were incubated at 37 ° C for 48 h (ICMSF, 1978). Lactic acid bacteria were enumerated on MRS agar (Oxoid) adjusted to pH 5.5 (Kitchell and Shaw, 1975), and incubated at 32°C for 3 days. Thermonuclease assay
Thermonuclease activity was determined by the quantitative procedure of Ibrahim (1981). Before testing, each supernatant was heated in a boiling bath for 15 min and its pH adjusted to 10. A standard curve was prepared using Micrococcal Nuclease (Sigma, 100/zmolar units/mg) and Salmon Sperm DNA (Difco). Detection of enterotoxins
Enterotoxins were detected using the single diffusion tube method as described by Kato et al. (1966), the double gel diffusion slide method proposed by Casman et al. (1969) and the sandwich ELISA technique of Fey et al. (1984). The reagents for ELISA test were obtained from W. Bommelli, Bern, Switzerland. Samples giving negative results with the microslide technique were concentrated by dialyzing against 30% (w/v) Carbowax 20M (Serva, Heildelberg, F.R.G.) and retested.
Results
The growth, enterotoxin production and thermonuclease activity of S. aureus strains FRI 137, FRI 361 and L 2 grown alone and in association with the starter culture are shown in Tables I, II and III, respectively. The presence of lactic acid bacteria had an inhibitory effect on the population of S. aureus but only after 18 h or more of incubation. The effect varied with the strain; after 48 h incubation, the percentages of inhibition were 18% (FRI 137), 11% (FRI 361) and 7% (L 2). Enterotoxin synthesis was strongly inhibited by the starter culture. When strain FRI 137 was grown in mixed culture, the level of enterotoxin C 1 at 18 h was 11% of the amount produced when the culture was grown alone; the corresponding figures for strains FRI 361 and L 2 (C 2 producers) were 20 and 31%, respectively. Furthermore, while enterotoxin C 1 was produced and accumulated during all phases of growth (pure and mixed culture), associative growth resulted in an apparent reduction of enterotoxin C 2 after 24-36 h incubation.
5.11 _+0.07 9.46_+0.03 9.49_+0.06 9.59_+0.05 9.28_+0.07 8.65-+0.05 7.78_+0.05
ND 0.13_+0.03 0.17_+0.04 0.25_+0.02 0.37+_0.08 0.39_+0.03 0.46_+0.05
Enterotoxin C 1 (p~g/ml) ND 2.36_+0.17 2.37+_0.19 2.36_+0.15 2.37_+0.15 0.16_+0.04 0.27+0.05
Thermonuclease (~g/ml)
6.96_+0.02 6.40_+0.03 6.00_+0.04 4.78_+0.03 4.81_+0.04 4.77_+0.03 4.81_+0.02
pH
4.81 +0.04 9.23_+0.05 9.70_+0.07 9.51_+0.02 9.57-+0.04 9.41_+0.07 8.70_+0.06
ND a 0.17_+0.03 2.0 _+0.1 2.6 +_0.2 2.9 _+0.4 6.3 +_0.3 10.5 _+0.6
4.78_+0.06 9.15_+0.03 9.68_+0.08 9.59_+0.03 9.46_+0.07 9.81_+0.04 9.78_+0.03
6.96_+0.04 6.49_+0.05 5.77_+0.03 4.79_+0.04 4.95_+0.06 4.67_+0.03 4.65+_0.04
logl0 S. aureus/ml
ND 0.006_+0.002 0.02 +0.005 0.03 - + 0 . 0 1 0.03 _+0.01 0.03 _+0.01 0.03 _+0.01
Mixed culture pH
lOglo S. aureus/ml
Thermonuclease (~g/ml)
Enterotoxin C 2 (/tg/ml)
Pure culture
ND, not detected.
0 8 12 18 24 36 48
Hour
ND 0.05_+0.02 0.30_+0.03 0.52_+0.09 0.50_+0.05 0.32_+0.07 0.15_+0.06
Enterotoxin C 2 (~g/ml)
ND 0.03 _+0 . 0 1 0.01 _+0.005 0.001 _+0.000 0.003_+0.001 0.02 _+0.007 0.05 _+0.01
Thermonuclease (/tg/ml)
6.92_+0.02 6.38_+0.03 5.70_+0.04 4.70_+0.02 4.84_+0.03 4.81_+0.05 4.86_+0.03
pH
Growth and synthesis of thermonuclease and enterotoxin C 2 by Staphylococc~" aureus FR1 361 in pure culture and in mixed culture with the starter HM1 (mean values-+ standard deviations)
TABLE II
6.96_+0.03 6.45_+0.04 6.01 _+0.02 5.12_+0.03 4.91_+0.04 4.68_+0.02 4.69_+0.03
ND " 0.32_+0.03 0.44_+0.04 2.2 _ + 0 . 2 11.8 _ + 0 . 3 14.3 _ + 0 . 4 23.1 _ + 0 . 3
5.00_+0.06 9.45_+0.02 9.75+-0.09 9.57+-0.06 9.67_+0.02 9.81_+0.05 9.53_+0.03
ND 2.37_+0.16 2.38_+0.15 2.36_+0.13 3.12+-0.20 7.05+_0.31 7.04_+0.23
Mixed culture pH
lOgl0 S. aureus/ml
Thermonuclease (/~g/ml)
Enterotoxin C 1 (/zg/ml)
Pure culture
lOglo S. aureus/ml
a ND, not detected.
0 8 12 18 24 36 48
Hour
Growth and synthesis of thermonuclease and enterotoxin C 1 by Staphylococcus aureus FRI 137 in pure culture and in mixed culture with the starter HM1 (mean values_+ standard deviations)
TABLE I
4.79+0.08 9.40+0.03 9.85-+0.07 9.70+0.06 9.63+0.04 9.99_+0.05 9.67+0.02
ND a 0.30_+0.04 0.44-+0.06 1.9 _+0.2 4.0 - + 0 . 3 4.4 _ + 0 . 2 7.2 +0.4
ND 0.21_+0.03 0.82-+0.08 0.81 _+0.07 0.82-+0.06 0.80_+0.08 0.81-+0.05
6.98+0.03 6.49_+0.06 5.70-+0.05 5.07_+0.05 4.85_+0.03 4.68_+0.04 4.75_+0.02
pH
4.88+0.04 9.48_+0.05 9.86_+0.06 9.56_+0.03 9.53_+0.07 9.53_+0.06 9.00_+0.03
log]0 S. aureus/ml
Thermonuclease (/Lg/ml)
log]o S. aureus/ml
Enterotoxin C 2 (~g/ml)
Mixed culture
Pure culture
a ND, not detected.
0 8 12 18 24 36 48
Hour
ND 0.11_+0.08 0.33_+0.06 0.58_+0.09 0.61_+0.06 0.76_+0.02 0.39_+0.08
Enterotoxin C 2 (~g/ml)
ND 0.09+0.01 0.80+0.06 0.81 +0.06 0.28_+0.03 0.27_+0.05 0.47+0.04
Thermonuclease (/t g/ml)
6.96+0.02 6.41+0.04 5.63-+0.03 4.97-+0.05 5.01_+0.02 5.00-t-0.03 5.05-+0.04
pH
Growth and synthesis of thermonuclease and enterotoxin C2 by Staphylococcus aureus L 2 in pure culture and in mixed culture with the starter HM1 (mean values _+standard deviations)
TABLE III
112
The effect of lactic acid bacteria on thermonuclease activity varied with the amount of enzyme produced in pure culture. With strains FRI 137 and L 2 (high producers of thermonuclease), a decrease between 18 and 36 h was observed, followed by an increase between 36 and 48 h. After 18 h incubation, the level of enzyme was always lower than that in pure culture. In contrast, at 8 and 48 h, thermonuclease activity of strain F R I 361 (low producer) appeared to be higher in associative culture than in pure culture. Inhibition of thermonuclease activity was observed between 12 and 36 h. Although the p H of mixed cultures decreased slightly faster, final values were lower in the pure cultures.
Discussion
The starter culture examined showed little inhibitory effect on growth of the three strains of S. aureus included which all attained population levels above ] 0 9 c f u / m ] in mixed cultures. Haines and H a r m o n (1973) found a stronger inhibitory influence of Streptococcus cremoris and Streptococcus lactis on growth of S. aureus strain F D A 243, which produces enterotoxin A. It should be noted that the slight inhibition observed in the present study seemed to occur in the stationary phase i.e. the exponential growth rate was not affected. This observation was also made by Haines and H a r m o n (1973). The effect of starter cultures on growth of S. aureus has been attributed, among other factors, to reduction of p H (Babel, 1977). However, the present results show that inhibition occurred in the late stages of incubation, when p H values of the mixed cultures were equal to or higher than those of pure cultures. Although substantial reduction in enterotoxins C 1 and C 2 were observed the starter culture used failed to prevent enterotoxin production. The ability of lactic bacteria and other organisms to prevent production of staphylococcal enterotoxins A and B in foods and broth has been reported by several authors (Haines and Harmon, 1973; Niskanen and Nurmi, 1976). The effect obtained apparently depends on the type and level of starter culture used as well as the sensitivity of the methods for enterotoxin detection. The inhibiton of production of enterotoxins C1 and C 2 and the decrease in enterotoxin C2 activity during the late stages of incubation in mixed culture may help to explain the low incidence of staphylococcal food-poisoning for Spanish cheeses prepared from sheep milk. In a previous work, 80% of S. aureus strains isolated from ovine mastitic milk in Spain produced enterotoxin C (Guti6rrez et al., 1982). Decrease in enterotoxin activity and even disappearance of toxins in the presence of lactic acid bacteria and other organisms have also been observed by Donnelly et al. (1968) in milk (enterotoxin A), Chordash and Potter (1976) in broth (enterotoxin A), Niskanen and Nurmi (1976) in dry sausage (enterotoxin C~) and Daoud and Debevere (1985) in broth and foods (enterotoxin A). The exact mechanism of the inhibitory effect on enterotoxin formation and the disappearance of toxins in mixed cultures is unknown. D a o u d and Debevere (1985)
113 suggested that it might be due to competition for essential nutrients, production of an inhibiting substance a n d / o r breakdown of the enterotoxin, and Chordash and Potter (1976) indicated that the decrease in enterotoxin A could be related to enzymes or other metabolites acting alone or assisted by a high acidity. One might conclude from the present results that, if lactic acid bacteria can inhibit the growth of pathogens in foods and decrease enterotoxin production, this is an additional desirable parameter when selecting and improving strains of microorganisms to be used for the fermentation of foods. In mixed culture, early decrease in thermonuclease activity could be due to proteolytic enzymes produced by lactic acid bacteria. Inactivation of thermonuclease by Streptococcus faecalis var. liquaefaciens was described by Lachica et al. (1972). Furthermore, Medwid and G r a n t (1980) reported that the above species produces a proteolytic substance called thermonuclease inactivating factor (TIF). Inactivation by Bacillus subtilis has also been reported (Daoud and Debevere, 1985). Our results show that the increase in thermonuclease activity during the late stages of incubation occurred when the colony-forming units of S. aureus had begun to decline. Since thermonuclease production was not observed when the starter culture was grown (without S. aureus) in APT broth at 3 0 ° C for 48 h (data not reported), it is unlikely that the increase could have been caused by the lactic acid bacteria. In associative culture, thermonuclease was detected whenever enterotoxin was detected ( > 1 n g / m l ) ; but the production of these metabolites did not parallel each other. This lack of correlation between the levels of enterotoxin and thermonuclease is consequence of the differences between strains of S. aureus in their ability to produce thermonuclease and enterotoxin. Also, the influence of lactic starter cultures varies with the strain used. Our results suggest that the thermonuclease test is a reliable indicator of S. aureus growth and enterotoxin production in associative cultures. However, this test cannot be used to predict either the number of S. aureus or the amount of enterotoxin accumulated.
Acknowledgements This work was supported in part by a grant (Project No. 4466-79) from the Spanish CAICYT, as well as by funds from Junta de Castilla-Lern (Consejeria de Educacirn y Cultura) and by D i p u t a c i r n Provincial de Lern. We are also indebted to Professor M.S. Bergdoll and to Miles-Martin, S.A.E. for providing enterotoxins and antisera, and the starter culture HM1, respectively.
References Babel, F.J. (1977) Antibiosis by lactic culture bacteria. J. Dairy Sci. 60, 815-821. Casman, E.P., Bennett, R.W., Dorsey, A.E. and Stone, J.E. (1969) The microslide gel double diffusion test for the detection and assay of staphylococcal enterotoxins. Health Lab. Sci. 6, 185-196. Chordash, R.A. and Potter, N.N. (1976) Stability of staphylococcal enterotoxin A to selected conditions encountered in foods. J. Food Sci. 41,906-909.
114 Daoud, S.M. and Debevere, J.M. (1985) The effect of Bacillus subtilis and Streptococcus faecalis var. liquaefaciens on staphylococcal enterotoxin A activity. Int. J. Food Microbiol. 2, 211-218. Donnelly, C.B., Leslie, J.E. and Black, L.A. (1968) Production of enterotoxin A in milk. Appl. Microbiol. 16, 917-924. Fey, H., Pfister, H. and Riiegg, O. 1984. Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C and D. J. Clin. Microbiol. 19, 34-38. Gilliland, S.E. and Speck, M.L. (1974) Antagonism of lactic streptococci toward Staphylococcus aureus in associative milk cultures. Appl. Microbiol. 28, 1090-1093. Guti&rez, L,M., Menes, I., Garcia, M.L., Moreno, B. and Bergdoll, M.S. (1982) Characterization and enterotoxigenicity of staphylococci isolated from ovine mastitic milk in Spain. J. Food Protect. 45, 1282-1289. Haines, W.C. and Harmon, L.G. (1973) Effect of selected lactic acid bacteria on growth of Staphylococcus aureus and production of enterotoxin. Appl. Microbiol. 25, 436-441. Ibrahim, G.F. (1978) Inhibition of Staphylococcus aureus under simulated Cheddar cheese making conditions. Austral. J. Dairy Technol. 33, 102-108. Ibrahim, G.F. (1981) A simple sensitive method for determining staphylococcal thermonuclease in cheese. J. Appl. Bacteriol. 51, 307-312. ICMSF (1978) Microorganisms in foods. 1. Their significance and methods of enumeration. University of Toronto Press, Toronto, 434 pp. Kato, E,, Khan, M., Kujovich, L. and Bergdoll, M.S. (1966) Production of enterotoxin A. Appl. Microbiol. 14, 966-972. Kitchell, A.K. and Shaw, B.G. (1975) Lactic acid bacteria in fresh and cured meats. In: J.G. Cart, C.V. Cutting and G.C. Whiting (Eds.), Lactic Acid Bacteria in Beverages and Foods, Academic Press, London, pp. 209-220. Lachica, R.V.F., Hoeprich, P.D. and Riemann, H.P. (1972) Tolerance of staphylococcal thermonuclease to stress. Appl. Microbiol. 23, 994-997. Medwid, R.D. and Grant, D.W. (1980) Inactivation of staphylococcal thermonuclease by an enzyme-linked factor produced by Streptococcus" faecalis var. liquaefaciens. J. Food Protect. 43, 201-202, 204. Niskanen, A. and Nurmi, E. (1976) Effect of starter culture on staphylococcal enterotoxin and thermonuclease production in dry sausage. Appl. Environ. Microbiol. 31, 11-20. Smith, J.L., Buchanan, R.L. and Palumbo, S.A. (1983) Effect of food environments on staphylococcal enterotoxins synthesis: A review. J. Food Protect. 46, 545-555.