J. appl. Bact. 1976,40, 115-117
Utilization of Amino Acids and Carbohydrates by Salmonella Serotypes at 22" and IT
JOHN LISTON AND PRINCE K. ZACHARIAH Institute for Food Science and Technology, College of Fisheries, University Seattle, Washington 98 I95, US.A.
of Washington,
Received 19 August 1975 and accepted 20 October 1975
ALTHOUGH SALMONELLAE have been shown to be capable of growth on seafoods at 8" (Matches & Liston, 1968), little is known concerning the nutritional determinants of their growth at low temperatures. Jezeski & Olsen (1961) showed that Pseudomonas spp. exhibited a preference for amino acids during growth at low temperature, and more recently it has been observed that they may exhibit an inductive shift to the synthesis of low temperature amino acid oxidases (Zachariah & Liston, in press). The purpose of this study was to determine whether salmonellae showed preference for and adaptation to amino acids during growth at low temperatures.
Materials and Methods Salmonella serotypes heidelberg, ATCC 8326; typlzimurium, ATCC 6994; and derby, ATCC 6966, were studied because they were among the serotypes most frequently isolated from human sources (Anon., 1965). The serotypes were cultured in nutrient broth (Difco) with 0.5% NaCl at room temperature (22") on a shaker for 18 h. Cells were harvested, washed and resuspended in peptone water blank to obtain lo4 cells/ml. An aliquot of 0.2 ml of this suspension was inoculated in sidearm flasks containing 20 ml basal medium (Zachariah & Liston, 1973) to which amino acids or carbohydrates at a concentration of 0.025 M were added. The flasks were then incubated on a shaker at 22" and 12"for about 140 h. Growth of these 3 Salmonella serotypes was determined by measuring absorbance at 660 nm wavelength.
Results All 3 strains grew well in casamino acids at 22" but only S. heidelberg grew at 12". No growth of any serotype tested was obtained at 22" or 12" when valine, leucine, glycine, or creatine were the sole carbon source, but aspartic acid and alanine supported growth of all 3 serotypes, but to a greatly differing degree at 22". Only S. heidelberg
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showed growth on alanine at 12". The lag period for growth at 12" by S. lieidelberg was greatly extended, even though the log phase was similar to that at 22", suggesting a possible induction process. All 3 serotypes grew in glucose and fructose at 22", but only S. heidelberg showed growth at 12". Ribose, sucrose and maltose also supported growth of all serotypes at 22", but none grew at 12". None of the serotypes was able to grow with citrate or lactose as sole carbon source. During the growth of S. heidelberg at 12" in the hexose medium, lag phase and log phase were greatly extended, suggesting a slow utilization of the substrate rather than a true low temperature adaptation. The extent of growth by S. heidelberg at the low temperature was less when sugar was the carbon source than when amino acids were used.
Discussion These results indicate the importance of nutrients in determining the ability of salmonellae to grow at low temperatures. Matches & Liston (1968) reported minimum temperatures for the serotypes used in this study, ranging between 5.5" and 6.8" when grown in trypticase soy broth. The same serotypes were also reported to grow on fish
TABLE 1
The efect of temperature and substrate on growth of Salmonella serotypes Absorbance at 660 nm after 72 h Temperature and substrates
S. typhimuriurn
S. derby
S. heidelberg
22" Aspartic acid Alanine Glucose Fructose
0.30 0.10 0.76 0.45
0.23 0.75 0.85 0.85
0.22 1.40 0.85 0.75
12" Aspartic acid Alanine Glucose Fructose
0.08 0.04 0.03 0.07
0.05 0.02 0.03 0.07
0.06 0.64 0.26 0.32
A
f
3
at 8" (Matches & Liston, 1968). Only S. heidelberg was able to grow at low temperature when carbon sources were restricted to amino acids or simple sugars, indicating a degree of nutritional versatility by this organism and a possible mechanism for adaptation to low temperature growth, particularly in the presence of certain amino acids. The inability of serotypes to grow at 12" on substrates which supported growth at 22" may indicate an increased nutritional requirement at the lower temperature or inability to use the substrate, due either to a failure of transport mechanisms or to a loss of enzyme activity. The technical assistance of Linda Shell is greatly appreciated. This investigation was supported by a grant from Food and Drug Administration 5R01 UI 00248-02 (formerly FD 00285-03).
SUBSTRATE UTILIZATION BY SALMONELLA
117
References ANON.(1965). Communicable Disease Center, Morbidity and Mortality Weekly Report 14, 210, U.S. Dept. of Health, Education and Welfare, Washington, D.C. JEZESKI, J. J. & OLSEN,R. H. (1961). The activity of enzymes at low temperatures. In Proc. Low Temp. Microbiof. Symp., New Jersey: Cainden, Campbell Soup Co. MATCHES, J. R. & LISTON,J. (1968). Growth of Salmonella on irradiated and non-irradiated seafoods. J. Fd Sci. 33, 406. ZACHARIAH, P. K. & LISTON,J. (1973). Temperature adaptability of psychrotrophic Pseuclomonas. Appl. Microbiol. 26, 437.
Utilization of Amino Acids and Carbohydrates by Salmonella Serotypes at 22" and IT
JOHN LISTON AND PRINCE K. ZACHARIAH Institute for Food Science and Technology, College of Fisheries, University Seattle, Washington 98 I95, US.A.
of Washington,
Received 19 August 1975 and accepted 20 October 1975
ALTHOUGH SALMONELLAE have been shown to be capable of growth on seafoods at 8" (Matches & Liston, 1968), little is known concerning the nutritional determinants of their growth at low temperatures. Jezeski & Olsen (1961) showed that Pseudomonas spp. exhibited a preference for amino acids during growth at low temperature, and more recently it has been observed that they may exhibit an inductive shift to the synthesis of low temperature amino acid oxidases (Zachariah & Liston, in press). The purpose of this study was to determine whether salmonellae showed preference for and adaptation to amino acids during growth at low temperatures.
Materials and Methods Salmonella serotypes heidelberg, ATCC 8326; typlzimurium, ATCC 6994; and derby, ATCC 6966, were studied because they were among the serotypes most frequently isolated from human sources (Anon., 1965). The serotypes were cultured in nutrient broth (Difco) with 0.5% NaCl at room temperature (22") on a shaker for 18 h. Cells were harvested, washed and resuspended in peptone water blank to obtain lo4 cells/ml. An aliquot of 0.2 ml of this suspension was inoculated in sidearm flasks containing 20 ml basal medium (Zachariah & Liston, 1973) to which amino acids or carbohydrates at a concentration of 0.025 M were added. The flasks were then incubated on a shaker at 22" and 12"for about 140 h. Growth of these 3 Salmonella serotypes was determined by measuring absorbance at 660 nm wavelength.
Results All 3 strains grew well in casamino acids at 22" but only S. heidelberg grew at 12". No growth of any serotype tested was obtained at 22" or 12" when valine, leucine, glycine, or creatine were the sole carbon source, but aspartic acid and alanine supported growth of all 3 serotypes, but to a greatly differing degree at 22". Only S. heidelberg
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J. LISTON AND P. K. ZACHARIAH
116
showed growth on alanine at 12". The lag period for growth at 12" by S. lieidelberg was greatly extended, even though the log phase was similar to that at 22", suggesting a possible induction process. All 3 serotypes grew in glucose and fructose at 22", but only S. heidelberg showed growth at 12". Ribose, sucrose and maltose also supported growth of all serotypes at 22", but none grew at 12". None of the serotypes was able to grow with citrate or lactose as sole carbon source. During the growth of S. heidelberg at 12" in the hexose medium, lag phase and log phase were greatly extended, suggesting a slow utilization of the substrate rather than a true low temperature adaptation. The extent of growth by S. heidelberg at the low temperature was less when sugar was the carbon source than when amino acids were used.
Discussion These results indicate the importance of nutrients in determining the ability of salmonellae to grow at low temperatures. Matches & Liston (1968) reported minimum temperatures for the serotypes used in this study, ranging between 5.5" and 6.8" when grown in trypticase soy broth. The same serotypes were also reported to grow on fish
TABLE 1
The efect of temperature and substrate on growth of Salmonella serotypes Absorbance at 660 nm after 72 h Temperature and substrates
S. typhimuriurn
S. derby
S. heidelberg
22" Aspartic acid Alanine Glucose Fructose
0.30 0.10 0.76 0.45
0.23 0.75 0.85 0.85
0.22 1.40 0.85 0.75
12" Aspartic acid Alanine Glucose Fructose
0.08 0.04 0.03 0.07
0.05 0.02 0.03 0.07
0.06 0.64 0.26 0.32
A
f
3
at 8" (Matches & Liston, 1968). Only S. heidelberg was able to grow at low temperature when carbon sources were restricted to amino acids or simple sugars, indicating a degree of nutritional versatility by this organism and a possible mechanism for adaptation to low temperature growth, particularly in the presence of certain amino acids. The inability of serotypes to grow at 12" on substrates which supported growth at 22" may indicate an increased nutritional requirement at the lower temperature or inability to use the substrate, due either to a failure of transport mechanisms or to a loss of enzyme activity. The technical assistance of Linda Shell is greatly appreciated. This investigation was supported by a grant from Food and Drug Administration 5R01 UI 00248-02 (formerly FD 00285-03).
SUBSTRATE UTILIZATION BY SALMONELLA
117
References ANON.(1965). Communicable Disease Center, Morbidity and Mortality Weekly Report 14, 210, U.S. Dept. of Health, Education and Welfare, Washington, D.C. JEZESKI, J. J. & OLSEN,R. H. (1961). The activity of enzymes at low temperatures. In Proc. Low Temp. Microbiof. Symp., New Jersey: Cainden, Campbell Soup Co. MATCHES, J. R. & LISTON,J. (1968). Growth of Salmonella on irradiated and non-irradiated seafoods. J. Fd Sci. 33, 406. ZACHARIAH, P. K. & LISTON,J. (1973). Temperature adaptability of psychrotrophic Pseuclomonas. Appl. Microbiol. 26, 437.
