The destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5°C and slow thawing at 4°C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5°C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at - 18°C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity

Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity (aw) was determined. The aw was adjusted by means of saturated salts with defined equilibrium relative humidity and the manure samples were stored aerobically at 20 degrees C. At aw levels higher than 0.93, a moderate increase in colony-forming units over 8-9 h was found for both strains; at aw levels of 0.89-0.75, there was a thousand-fold reduction. Extended storage resulted in a million-fold reduction of Salmonella enteritidis in 8 days at an aw of 0.89. At higher and lower levels of aw, the reduction was less extensive.     

Biofilm formation by different strains of Salmonella typhimurium in artificial systems

The ability of 14 different strains of Salmonella typhimurium to biofilm formation depending on genotype and culture conditions was investigated in artificial systems: in 96-well plastic microtitre plates, plastic and glass tubes, plastic Petri dishes and on microscope glasses. Quantitative biofilm growth was monitored by using an assay based on crystal violet staining, while planctonic growth in the same cultures was monitored by absorbance in iEMS Reader MF, and qualitatively--by digital photo and visually. Optimal rate between growth and biofilm indications for all strains was determined at initial cell concentration 10(6-7) KOE/ml and culture incubation at t degrees 28 degrees C. The nutrient content of the medium significantly influenced the quantity of produced biofilm. The nutrient broth LB without NaCl was more effective in promoting biofilm formation, than LB itself. The least quantity of biofilm was formed in water. The genotype of the strains also critically influenced the quantity of produced biofilm. Nonmotile mutants cells had reduced ability to form biofilm. RpoS mutant cells produced significantly less biofilm as compared with cells of isogenic parent strains. The chemical content of plastic and glass also influenced biofilm formation.     

Interferon enhances the bactericidal activity of peritoneal exudate macrophages against Salmonella typhimurium in mice

The treatment of macrophages of mouse peritoneal exudate has been found to enhance their bactericidal activity with respect to S. typhimurium. This activation depends on the dose of interferon and the cells/bacteria ratio. The action of interferon is species-specific.     

Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of Salmonella typhimurium termed A1-R. The present report demonstrates that S. typhimurium A1-R destroys tumor blood vessels and this is enhanced in tumors with high vascularity. Red fluorescent protein (RFP)-expressing Lewis lung cancer cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin and footpad of nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Color-coded in vivo imaging demonstrated that the LLC-RFP ear tumor had the highest cell density and the footpad tumor had the least. The ear tumor had more abundant blood vessels than that on the back or footpad. The tumor-bearing mice were treated with A1-R bacteria via tail-vein injection. Tumors in the ear were the earliest responders to bacterial therapy and hemorrhaged severely the day after A1-R administration. Tumors growing in the back were the second fastest responders to bacterial treatment and appeared necrotic 3 days after A1-R administration. Tumors growing in the footpad had the least vascularity and were the last responders to A1-R. Therefore, tumor vascularity correlated positively with tumor efficacy of A1-R. The present study suggests that bacteria efficacy on tumors involves vessel destruction which depends on the extent of vascularity of the tumor.Key words: tumor targeting bacteria, Salmonella typhimurium A1-R, Lewis lung carcinoma, RFP, GFP, nestin, nude mice     

The mosaic of proteins synthesized by Salmonella typhimurium

A profusion of proteins with heterologous serological specificities was synthesized by S. typhimurium grown on artificial media; accordingly, sera prepared in rabbits with these proteins displayed an abundance of antibodies reacting, in agar gel, against numerous heterologous proteins. the absorption of the sera with different Enterobacterial proteins proved that the S. typhimurium proteins are a mixture of specific proteins, and common E. coli and Salmonellae determinants; in addition, a group of strongly cross-precipitating proteins common to S. typhimurium and S. choleraesuis and to S. typhimurium and S. kentucky were identified that were not present in the proteins common to S. enteritidis, S. typhi and E. coli, or in the S. paratyphi A proteins used absorptions. The specific proteins of S. typhimurium were synthesized on artificial media in, apparently, smaller amounts than the common proteins; their role in the protection of mice against infection with their natural pathogen was, however, proof of their specificity and contrasted with the ineffectiveness, in protecting the mice, of the common proteins.     

His+ reversions caused in Salmonella typhimurium by different types of ionizing radiation

The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co gamma rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV alpha particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/micron. The ratio of the effectiveness of this radiation to gamma rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the alpha particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames [Mutat. Res. 113, 173-215 (1983)] for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study [Roos et al., Radiat. Res. 104, 102-108 (1985)] greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating.     

Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of Salmonella typhimurium termed A1-R. The present report demonstrates that S. typhimurium A1-R destroys tumor blood vessels and this is enhanced in tumors with high vascularity. Red fluorescent protein (RFP)-expressing Lewis lung cancer cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin, and footpad of nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Color-coded in vivo imaging demonstrated that the LLC-RFP ear tumor had the highest cell density and the footpad tumor had the least with the ear tumor having more abundant blood vessels than that on the back or footpad. The tumor-bearing mice were treated with A1-R bacteria via tail-vein injection. Tumors in the ear were the earliest responders to bacterial therapy and hemorrhaged severely the day after A1-R administration. Tumors growing in the back were the second fastest responders to bacterial treatment and appeared necrotic 3 days after A1-R administration. Tumors growing in the footpad had the least vascularity and were the last responders to A1-R. Therefore, tumor vascularity correlated positively with tumor efficacy of A1-R. The present study suggests that bacteria efficacy on tumors involved vessel destruction which depends on the extent of vascularity of the tumor.     

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.     

Sterol hydroperoxide metabolism by Salmonella typhimurium

In order to rationalize multiphasic dose-response data evincing mutagenicity towards Salmonella typhimurium TA1537 for sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide their metabolism by the bacterial test strain was investigated. The 5 alpha-hydroperoxide was isomerized to the 7 alpha-hydroperoxide and reduced to 5 alpha-cholest-6-ene-3 beta,5-diol; the 7 alpha-hydroperoxide was reduced to cholest-5-ene-3 beta,7 alpha-diol and transformed to 3 beta-hydroxycholest-5-en-7-one. The 3 beta,5 alpha-diol and 3 beta,7 alpha-diol were not interconverted nor was either transformed to the 7-ketone.     

Determining specific growth rates in different regions of Salmonella typhimurium colonies

Computer-aided image analysis (IA) techniques were used to determine specific growth rates (μ) in different regions of Salmonella typhimurium colonies grown on solid medium at 30°C. Captured transmission images were converted to optical density values, allowing calculation of μ. Data show that growth was exponential throughout the colony during the period studied (between 13 and 21 h after inoculation), but that μ varied from 0·69 to 0·37 h⁻¹ at the periphery and centre, respectively. Results demonstrate regional variation in μ values within colonies, and support conceptual models of colony development. The technique described illustrates that kinetic growth data can be derived within bacterial colonies, and this may lead towards an improved understanding of the growth dynamics of these structures.     

Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium

The effect of a high hydrostatic pressure treatment on the Gram-positive Listeria monocytogenes strain Scott A and the Gram-negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions. Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5.6) and sodium phosphate (pH 7.0) suspension buffers. Increasing pressure treatments resulted in an exponential decrease of cell counts. Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments. Progressive morphological changes were evident with the pressure increase. Cell lysis only appeared with the highest pressure treatments. Cell volume was not affected by pressure treatment. A progressive decrease of deltapH (pHin - pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase. A parallel lowering of membrane potentials was measured.     

Methionine synthesis by extracts of Salmonella typhimurium

1. Following the genetic studies by Smith (1961) and Smith & Childs (1963) with methionine auxotrophs of Salmonella typhimurium, methionine formation from homocysteine has been investigated with cell-free extracts of this organism. 2. As found with Escherichia coli (Woods, Foster & Guest, 1964), methyl groups are formed by an N(5)N(10)-methylenetetrahydrofolate reductase. They are then transferred to homocysteine by either a simple N(5)-methyltetrahydropteroyl-triglutamate-homocysteine methyltransferase or alternatively a cobalamin-dependent N(5)-methyltetrahydrofolate-homocysteine methyltransferase. 3. S. typhimurium differs from E. coli in being able to synthesize significant amounts of cobalamin.     

Metabolism of 1,8-dinitropyrene by Salmonella typhimurium

Earlier work has shown that many nitroaromatic and nitroheterocyclic compounds are directly 'activated' to their ultimate mutagenic forms through the action of bacterial nitroreductase enzymes. However, in the case of 1,8-dinitropyrene (DNP) and certain other nitroarenes the pathway of activation is more complex and neither the identity of the ultimate mutagens nor the nature of the DNA adducts formed are known. We now show that Salmonella typhimurium strains TA98 and TA1538, which are sensitive to DNP and have wild type nitroreductase complements, do metabolize DNP to 1-amino-8-nitropyrene (ANP) and 1,8- diaminopyrene (DAP) but that these compounds are much weaker mutagens than DNP. These two strains (TA98 and TA1538) contain two separable components of nitroreductase activity as determined using nitrofurazone as the substrate. The major component, at least, is capable of reducing both 1-nitropyrene (NP) and DNP although the rates are much lower than with nitrofurazone. TA98NR , a mutant of TA98 that is resistant to nitrofurazone and NP but not to DNP, lacked the major nitroreductase but retained two minor components. In contrast, a mutant ( DNP6 ) which is resistant to DNP (but not to NP) contained a full complement of nitroreductases. When the metabolism of [3H]DNP by crude extracts of TA98 was re-examined, previously undetected metabolites were found. These were more polar than DAP and ANP and were also seen when TA98NR was used as the source of enzyme. These metabolites were not formed when enzymes from TA98DNP6 or TA98NR / DNP6 were used. This work supports the notion that some enzymic activity other than (or in addition to) nitroreductase is required for the activation of DNP and that the new polar metabolites may be related to this process.