Comparison of Methods for the Isolation of Salmonella from Bone Meal

The efficiency of some methods for the isolation of Salmonella from bone meal was studied. Grinding of the granulated, industrial product was found to increase Salmonella recovery. An improvement in the efficiency of the pre-enrichment method was obtained by adding deoxycholate (1%) to mannitol broth. Selective enrichment in selenite cystine medium gave better results than in Kauffmann-Müller medium, when small inocula were used. Experimental data showed that, in the presence of large numbers of coliforms and very small numbers of Salmonella, the Kauffman-Müller medium offers better results, but, in the presence of smaller numbers of coliforms, selenite cystine is preferable. As solid selective medium, Brilliant Green Agar gave, in our laboratory, better results than S S medium.     

Sensitivity and Specificity of Different Methods for the Isolation of Salmonella from Pigs

Three different selective enrichment media, Rappaport-Vassiliadis broth (RV), selenite broth (SB) and Müller-Kauffmann tetrathionate broth (MKTB), in combination with plating on modified brilliant green agar (BGA), were compared for the isolation of Salmonella from samples of pig feces. These conventional methods were also compared with a new ELISA kit in conjunction with RV and SB enrichment. Of the conventional methods, enrichment in RV had a higher sensitivity and selectivity than SB and MKTB. Recovery of S. typhimurium from MKTB was significantly poorer than recovery of other serotypes. The combination of RV enrichment and ELISA was as good as the conventional method involving RV enrichment, with a similar high sensitivity and specificity.     

Technique for the Isolation of Ribonucleic Acid-Rich Mutants from Escherichia coli

A technique for the isolation of ribonucleic acid (RNA)-rich mutants of Escherichia coli is described. Mutagenized cells were centrifuged to isopycnic equilibrium on potassium tartrate or cesium sulfate gradients. Samples from the region of the gradients slightly denser than the majority of the cells were spread on agar plates, and the resulting clones were tested for increased RNA to protein ratios.     

Isolation of a cytotoxin from L-form Salmonella typhimurium

Abstract A cytotoxin protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 μg/ml, with a complete lysis obtained at 5 μg/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1–0.5 μg/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor α. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 μg/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.     

Isolation and characterization of the aadA aminoglycoside-resistance gene from Salmonella choleraesuis

The streptomycin- and spectinomycin-resistance gene of Salmonella choleraesuis was cloned and its nucleotide sequence determined. The gene is 789 bases long, encoding a protein of a predicted size of 29,353 Da. The gene product inactivated streptomycin and spectinomycin by an adenylation modification. It is homologous (c. 40% total identity) to streptomycin adenylyltransferase, a 3'(9)-O-nucleotidyltransferase (AAD(3')(9)), which is encoded by the aadA gene in Escherichia coli, Agrobacterium tumefaciens, Klebsiella pneumonia, and Serratia marcescens. The AadA protein of S. choleraesuis differs significantly from the other AadA proteins, indicating that it may have diverged from the other members of this family earlier in evolution. Southern hybridization analysis revealed that homologous aadA sequences were also present in other streptomycin-resistant Salmonella species.     

Isolation and characterization of Salmonella enterica from Antarctic wildlife

In recent years, the human presence in Antarctica has increased and as a consequence, the possibility of microorganisms’ introduction. The aims of this work were to determine the presence of Salmonella enterica in Antarctic seabirds and sea mammals, to characterize the isolates identified, and to determine the genetic relation of Antarctic S. enterica isolates among them and compare with isolates of human, animal, and food sources recovered in Argentina. During the summer 2000 and 2002 in Potter Peninsula, and during the summer 2001 and 2003 in Hope Bay, a total of 1,739 fecal samples from Antarctic animals were collected and analyzed. In summer 2000, S. Newport and S. Enteritidis were isolated from 8.9% of southern giant petrels (Macronectes giganteus). In summer 2003, S. Enteritidis was isolated from 1.5% of Adelie penguins (Pygoscelis adeliae), from 5.5% of skuas (Stercorarius sp.), from 5.4% of kelp gulls (Larus dominicanus), and from 5.6% of Weddell seals (Leptonychotes weddelli). All the isolates belonging to the same serovar showed indistinguishable genomic profiles by Pulse-Field Gel Electrophoresis (PFGE) with XbaI and BlnI restriction enzymes and by Random Amplified Polymorphic DNA (RAPD-PCR). In addition, these Antarctic strains were different from S. enterica isolates from different sources identified in Argentina during the same or close time periods.     

Isolation and crystallization of unadenylylated glutamine synthetase from Salmonella typhimurium

The enzyme glutamine synthetase (GS) has been isolated from a mutant strain of Salmonella typhimurium, constructed by Kustu, which lacks the enzymatic activity for adenylylation of glutamine synthetase. Thus the purified GS is uniformly unadenylylated, as confirmed by gel electrophoresis and enzyme assays. It crystallizes readily in many morphologies, at least six of which are distinct polymorphs. The most favorable crystal form for structural studies belongs to space group C2, with unit cell dimensions a = 235.5 A, b = 134.5 A, c = 200.1 A, beta = 102.8 degrees, and with one GS molecule per asymmetric unit. The crystals diffract to about 2.8 A resolution in rotation X-ray photographs and thus appear suitable for structural studies at moderate resolution. These crystals are isomorphous with crystalline GS from Escherichia coli in both adenylylated and unadenylylated states, suggesting that the enzymes from the two bacteria are similar molecules, and that adenylylation does not greatly affect the conformation of the molecule.     

Isolation of a mutant from Salmonella typhimurium producing acyl-deficient lipopolysaccharides

The present paper describes the isolation and characterization of a mutant (mutant Ts7) of Salmonella typhimurium that is conditionally defective in the incorporation of dodecanoic and tetradecanoic acid into lipopolysaccharide precursor structures. Enrichment of mutant Ts7 was achieved by free-flow electrophoresis and was based on a previous observation that at least some Salmonella mutants conditionally blocked in the synthesis of the 3-deoxy-D-manno-octulosonic acid (dOc1A)-lipid-A region exhibit higher electrophoretic mobilities than cells with intact dOc1A-lipid-A regions. Under nonpermissive conditions (42 degrees C) mutant Ts7 accumulates at least two incomplete dOc1A-lipid-A structures. One is made up of glucosamine, phosphate, dOc1A, and 3-hydroxytetradecanoic acid in a molar ratio 1.0:1.3:1.0:2.2 and is devoid of dodecanoic and tetradecanoic acid. The other structure has the same basic structure but contains hexadecanoic acid.     

Isolation of bacteriophage 14-lysogenized Salmonella from the freshwater aquarium snail Apullaria.

A naturally occurring Salmonella mikawasima serologically converted by phage 14 (6,7,14:y:e,n,z15) has been isolated for the first time. An S. tennessee variant seroconverted by phage 14 (6,7,14:z29:-) was also isolated. The source of these salmonellae was the common freshwater aquarium snail Ampullaria. Phage 14 prepared from these serovariants was lytic for S. bovis-morbificans (6,8:r:1,5) and for S. hadar (6,8:z10:e,n,x).