Differences in initial rate of intracellular killing of Salmonella typhimurium by resident peritoneal macrophages from various mouse strains

To determine the underlining mechanism of the difference in innate susceptibility of mouse strains to infection by Salmonella typhimurium, the ingestion and in vitro intracellular killing of S. typhimurium by resident peritoneal macrophages of mouse strains that differ in natural resistance to this microorganism has been studied. The results revealed that the rate constants of in vitro phagocytosis (Kph) in the presence of inactivated rabbit immune serum did not differ between macrophages of susceptible C57BL/10 and resistant CBA mice (for both strains: Kph = 0.021 min-1). The rate constant of in vitro intracellular killing (Kk) was determined 1) after in vivo phagocytosis (CBA, Kk = 0.055 min-1; C57BL/10, Kk = 0.031 min-1), 2) after in vitro phagocytosis of preopsonized bacteria (CBA, Kk = 0.020 min-1; C57BL/10, Kk = 0.012 min-1), and 3) during continuous phagocytosis in vitro (CBA, Kk = 0.029 min-1; C57BL/10, Kk = 0.013 min-1). With all three approaches, the initial rate of intracellular killing by normal macrophages of Salmonella-resistant CBA mice amounted to about 1.7 times the value found for macrophages of susceptible C57BL/10 mice (p less than 0.01). This trait difference was independent of the previous way of ingestion of the bacteria, unaffected by the kind of opsonization, and specific for S. typhimurium, because Staphylococcus aureus and Listeria monocytogenes were killed by macrophages of these mouse strains with equal efficiency (p greater than 0.50). These findings indicate that a difference in genetic background expressed in the efficacy of intracellular killing by resident peritoneal macrophages immediately upon ingestion of S. typhimurium is relevant for the innate resistance of mice against S. typhimurium.     

Genetic mapping of the Salmonella typhimurium pepB locus.

Transposon technology has been used to map the pepB locus of Salmonella typhimurium. This locus is cotransducible by phage P22 with glyA and strB at min 56 on the Salmonella genetic map. The gene order is strB pepB glyA.     

Mapping of the hemE locus in Salmonella typhimurium.

A new type of heme-deficient mutant was isolated in Salmonella typhimurium by neomycin selection. The mutant was deficient in uroporphyrinogen decarboxylase activity, coded by the hemE gene. The hemE gene was located between the genes rif and thi at 128 min on the chromosomal map of S. typhimurium.     

Genetic mapping of the Salmonella typhimurium pncB locus.

The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative to pyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.     

The effect of step changes in sucrose concentration on the growth of Salmonella typhimurium LT2

Water activity is a method of preservation that can affect microbial growth in foods and that may fluctuate during their processing, distribution and storage. Sucrose has been used to change the water activity of microbiological culture media. Suspensions of Salmonella typhimurium LT2 in the exponential phase of growth have been subjected to step changes in sucrose concentration at 20°C. The changes in the numbers of viable bacteria were measured with time and the experimental growth curves compared with predictions based on growth data obtained at constant sucrose concentrations. Steps down in sucrose concentration showed some apparent loss of viability after the step followed by growth at a rate close to the expected value. Steps up in sucrose concentration resulted in a greater apparent loss of viability after the step and either growth or the inducement of lag, depending on the final concentration of sucrose. A series of small steps up in sucrose concentration to 45% (w/v) was able to sustain growth where it was not possible by inoculation directly into this concentration. Improved recovery of bacteria subject to osmotic stress was possible with a medium containing sodium chloride.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: Ity gene is expressed in vivo by 24 hours after infection

The early response of inbred mice to infection with S. typhimurium is controlled by the mouse Chromosome 1 locus, Ity. To better understand the expression of this gene, the initial interactions between the reticuloendothelial system (RES) and i.v. injected salmonellae were compared in resistant (Ityr) and susceptible (Itys) mice. In both mouse strains 99% of the bacteria was cleared from the blood within 2 hr, and uptake of S. typhimurium by splenic and hepatic macrophages was similar regardless of Ity genotype. In vivo phagocytosis of bacteria was followed by a 30 to 60% decline in viable bacteria, which was attributed to the bactericidal activity of RES macrophages. Experiments with radiolabeled S. typhimurium strains TML and TML/TS27 (a temperature-sensitive mutant) confirmed that the efficiency of this early phase killing was not under Ity control. Despite the equivalent uptake and initial bactericidal activity by resident macrophages, bacterial numbers in the RES organs of Itys mice were significantly greater than in Ityr mice by approximately 24 hr after infection. These data suggest that Ity regulates the level of surviving intracellular bacteria that accumulate within resident macrophages of the liver and spleen.     

Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa

The bacteriostatic effect of methioninyl adenylate (MAMP)—a specific inhibitor of the enzyme methionyl-tRNA synthetase—was investigated on Salmonella typhimurium and Pseudomonas aeruginosa.0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1–2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P. fluorescens and P. putida.The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the same concentration of the inhibitor. — P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture.—MAMP has no effect on P. alcaligenes.The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNA^met from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5phosphodiesterase which degrades MAMP is 10–20 fold higher in the second than in the first species.Non Standard Abbreviations MAMP Methioninyl adenylate - MTS methionyl-tRNA synthetase (EC 6.1.1.10) - VTS valyl-tRNA synthetase (EC 6.1.1.0) - Tris trihydroxymethylaminomethane Dedicated to Prof. R. Y. Stanier on the occasion of his 60th birthday     

Mutagenic effect of nialamide on Salmonella typhimurium

The mutagenicity of an antidepressant drug, nialamide, was studied with Salmonella typhimurium TA1535-8. Nialamide was mutagenic for strain TA1535 in the absence of rat liver extracts.     

The effect of sodium chloride concentration and pH on the growth of Salmonella typhimurium colonies on solid medium

The growth of Salmonella typhimurium colonies on a model food system (agar solidified culture medium) was followed. Colony radius, determined using computer image analysis (IA) techniques, and viable cell number per colony were measured as indices of colony growth, and the effect of [NaCl] (0.5–3.5% (w/v)) and pH (7.0–5.0) on colony growth at 30°C was observed; colonies were point inoculated from serial dilutions. Colony growth (between 13 and 26 h after inoculation) was linear when expressed in terms of radius, and exponential when expressed in terms of viable cell number per colony. Overall, both increasing the [NaCl] and decreasing the pH had little effect on colony growth, other than to delay the onset of linear radial growth. Initial specific growth rate (μ) ranged from 0.73 to 0.87 h⁻¹. Thin films of agar medium on microscope slides allowed the growth of microcolonies to be observed after just 4 h incubation. A greater understanding of the growth kinetics of bacterial colonies, and the effects of environment on such data, may enable better control of foodborne bacterial pathogens, and consequently an improvement in food product safety.     

Thymidine-requiring mutants of Salmonella typhimurium that are defective in deoxyuridine 5''-phosphate synthesis.

In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.     

The effect of transient temperatures on the growth of Salmonella typhimurium LT2. II: excursions outside the growth region

The effect of fluctuating temperatures on microbial growth is important in the passage of foods from production to consumption. Suspensions of Salmonella typhimurium have been subjected to sinusoidally time-varying temperatures of periods from 60 to 240 min between 4°C and 22°C, that is within and below the growth temperature range. The suspensions were prepared with two concentrations of sodium chloride and adjusted to two different values of pH. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. Generally, the experimental average generation times exceeded the predictions by not more than 10% In enumerating viable bacteria in the suspensions containing 3.5% (w/v) sodium chloride it was necessary to use sodium chloride in the diluent and recovery medium in order to recover the bacteria quantitatively.     

The effect of transient temperatures on the growth of Salmonella typhimurium LT2. I: cycling within the growth region

The effect of fluctuating temperatures on microbial growth is important in the passage of foods through the food chain. Suspensions of Salmonella typhimurium were subjected to sinusoidally time-varying temperatures of periods from 40 to 480 min within their growth temperature range. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. The experimental average generation times exceeded the predictions by less than 30%, although the discrepancy increased with cycle frequency. Instantaneous growth rates obtained for the 480 min cycles were in agreement with those predicted from isothermal behaviour.     

Fine-structure genetic map of the cysB locus in Salmonella typhimurium.

A genetic map of the cysB region of the Salmonella typhimurium chromosome was constructed using bacteriophage P22-mediated transduction. Strains bearing delta (supX cysB) mutations were employed to divide this regulatory locus into 12 segments containing a total of 39 single-site mutations. Twenty-five of these single-site mutations were further ordered by reciprocal three-point crosses. The results do not support the concept of multiple cistrons at cysB and suggest that the abortive transductants previously observed in crosses between certain cysB mutants were due to intracistronic complementation. The prototrophic cys-1352 mutation, which causes the constitutive expression of the cysteine biosynthetic enzymes, was found to lie within the cysB region itself. It is bracketed by mutations, which lead to an inability to derepress for these enzymes and result in auxotrophy for cysteine.