A rat model of infection by Salmonella typhimurium or Salm. enteritidis

Salmonellosis in the rat has many similarities with the disease in humans, with the ileum thought to be the main site of colonization/invasion in both species. Thus, the rat may be a useful way to study the mechanism of infection by these pathogenic bacteria. A series of infection trials carried out with Hooded Lister rats showed that a salmonella infection persisted for an extended period of time and that salmonellae bind to the small intestinal epithelium as early as 4 h after intragastric intubation. Reinfection from the large intestine may not therefore initially play a significant role in the salmonella infection process. The rat model may therefore provide a means to test in vivo interventionist strategies, designed to block binding of the pathogens in the gastrointestinal tract.     

Survival of Salmonella enteritidis PT4 and Salm. typhimurium Swindon in aerosols

A.S. McDERMID AND M.S. LEVER. 1996. Small particle aerosols of plate-grown Salmonella enteritidis and Salm. typhimurium were generated and maintained within a rotating drum at 75% relative humidity and 24°C for 2 h. Plate-grown organisms were found to be more aerosol-stable than broth-grown organisms. Differences were observed between the two species; plate-grown Salm. typhimurium retained 100% viability after 2 h compared to approximately 70% for plate-grown Salm. enteritidis . A large proportion of cells of both serotypes remained viable in aerosols after 2 h, confirming the potential for airborne transmission for these organisms, e.g. within henhouses and during food     

Rapid identification of Salmonella typhimurium, S. enteritidis and S. virchow isolates by polymerase chain reaction based fingerprinting methods.

In this study we used and evaluated three rapid molecular typing methods for the identification of three frequent, clinically significant Salmonella serovars on the basis of the ease, simplicity and reproducibility of the chosen methods. We determined the genetic diversity among several isolates of Salmonella enteritidis, S. typhimiurium and S. virchow, and compared them with other enterobacteria by using the repetitive extragenic palindromic (REP) sequences, the enterobacterial repetitive intergenic consensus (ERIC) sequences, and the 16S-23S rDNA intergenic spacer region (ITS 1). The objective was to evaluate their potential application to discriminate among members of the species Salmonella enterica subspecies enterica using the genetic diversity of the group found by genomic fingerprinting. The three different serovars of Salmonella studied gave reproducible and distinguishable profiles using whichever of the above mentioned polymerase chain reaction (PCR) methods assayed. The conserved patterns in each serovar allowed for easy differentiation from other serovars of Salmonella.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

Starvation survival of Salmonella enteritidis.

Washed cells of Salmonella enteritidis harvested from a defined medium during logarithmic growth were subjected to starvation in pH 7 phosphate buffer at 37 C. Viability was measured by slide cultures and plate counts. The survival of cell suspensions equivalent to 1 to 10 mg (dry wt)/ml was influenced by cryptic growth. The rate of cryptic growth, assessed by plate counts, increased with cell density and could not be alleviated by starvation with dialysis. Dialysis of the starving culture did retard the onset of cryptic growth but did not eliminate it, indicating that the major substrates for regrowth were relatively large cellular components. In phosphate buffer, 6.7 homologous heat-killed cells allowed for the doubling of one S. enteritidis cell. Cryptic growth was not observed when cells were starved on the surface of membrane filters or in suspensions equivalent to 20 mug (dry wt)/ml (105 cells/ml). Similar half-life survival times were calculated for both these populations, but the shape of their survival curves differed significantly. These differences were attributed to stress factors encountered during cell preparation and during starvation. The half-life survival time of S. enteritidis starved at 20 mug (dry wt)/ml was 140 h in phosphate buffer, 82 h in 3,6-endomethylene-1,2,3,-6-tetrahydrophthalic acid buffer, and 77 h in tris(hydroxymethyl)aminomethane buffer.     

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64·9% of environmental isolates. The E/NF system gave a positive identification to 88·0% of isolates and the 20E to 79·5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.     

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enteritidis.

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enterica serovar Enteritidis (S. enteritidis) is described. The procedure consists of plasmid DNA purification, its digestion with restriction endonuclease TaqI, electrophoresis, charge-coupled device camera scanning of the gels, and an analysis of the restriction patterns with the software Gel Manager. The system allowed us to analyse, in detail, results of plasmid profiling in more than 600 field strains of S. enteritidis. In addition to plasmid-free and virulence plasmid only containing strains, 15 additional plasmid types were detected. All the images and detailed protocols are available at the Web site http://www.clark.cz/vri/salmon.htm.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

Plasmid characterization and pulsed-field electrophoretic analysis demonstrate that ampicillin-resistant strains of Salmonella enteritidis phage type 6a are derived from Salm. enteritidis phage type 4

Plasmid incompatibility studies have demonstrated that strains of Salmonella enteritidis phage type (PT) 6a resistant to ampicillin possess a 36 megadalton incompatibility group (Inc) X plasmid coding for resistance to ampicillin which is capable of converting strains of Salm. enteritidis belonging to PTs 1 and 4 to PT 6a, and PT 8 to PT 13. However, pulsed-field gel electrophoresis (PFGE) has demonstrated that all clinical isolates of PT 6a have a characteristic Xba I pulsed-field profile which is distinct from that of PT 1 and which can only be differentiated from that of PT 4 by the presence of plasmid-associated fragments of less than 45 kb. It is concluded that ampicillin-resistant strains of Salm. enteritidis PT 6a are derived from strains of Salm. enteritidis PT 4 by acquisition of an Inc X ampicillin resistance plasmid.     

Transformation of Salmonella typhimurium with plasmid DNA: differences between rough and smooth strains.

Lipopolysaccharide-defective mutants of Salmonella typhimurium were transformed by plasmid DNA with a Ca2+ treatment method. Only those mutants with an Rc or Rd2 chemotype, due to galE or rfaF mutations, respectively, gave efficiencies greater than 10(5) transformants per microgram of DNA, frequencies 8- to 630-fold higher than with smooth strains or other rough mutants.     

Intra-strain heterogeneity in expression of lipopolysaccharide by strains of Salmonella virchow

Strains of Salmonella virchow express long-chain lipopolysaccharide which migrates as one of two distinct profiles during SDS-PAGE. Both LPS phenotypes were detected within a given strain of Salm. virchow and shared the O = 7 antigen.     

Utilization of D-xylose by wild-type strains of Salmonella typhimurium.

Enzyme studies of strains of Salmonella typhimurium representing biotypes that utilized D-xylose rapidly (xylose strong) or slowly (xylose weak) showed that they were different in the utilization of D-xylose because the xylose-weak strains were deficient in the transport of D-xylose. This observation is consistent with the idea that strains of the different xylose-weak biotypes, e.g. biotypes 17 to 32, were descended from strains of xylose-strong types, particularly from biotype 1.     

Egg contamination by Salmonella serovar enteritidis following vaccination with Delta-aroA Salmonella serovar typhimurium

The efficacy of an aroA Salmonella serovar typhimurium modified live vaccine to decrease internal egg contamination after oral challenge of hens with egg-contaminating Salmonella serovar enteritidis was assessed. Challenge was with a mixed phenotype of S. enteritidis that had virulence characteristics previously associated with enhanced oral invasiveness and egg contamination in chickens. Immunized birds had fewer positive ovary/oviduct pools and lower cfu g(-1) cecal contents than did non-immunized birds, but the differences were not significant. The number of positive intestinal (duodenum, jejunum, ileum) and organ (spleen, kidney, liver) pools following challenge from each treatment group were equivalent. Most importantly, immunization did not decrease egg contamination. These results suggest that the ability of modified live vaccines to reduce internal egg contamination by S. serovar enteritidis can be assessed using characterized strains for challenge.     

Spontaneous, ultraviolet and ionizing radiation mutagenesis in two auxotrophic strains of Salmonella typhimurium carrying an R plasmid.

Ultraviolet-induced, gamma-induced and spontaneous mutation yields were studied in two different auxotrophic strains of Salmonella typhimurium in the presence and absence of the UV-protecting drug resistance transfer factor R-Utrecht. One strain, carrying the hisC527 (amber) mutation, showed significantly increased spontaneous, UV- and gamma-induced mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R factor, but appeared to show no significant increase in spontaneous mutability and only a very slight increase in gamma-mutability when carrying the R factor. These results demonstrate that the R-Utrecht plasmid, known to enhance UV-induced mutation yields in S. typhimurium, can also significantly enhance both spontaneous and gamma-induced mutation yields in this species. The latter effects are not so discernible with all markers, however, as shown by the results with strains carrying the trpD1 mutation. Enhancement of spontaneous mutability thus appears to be correlated with enhancement of gamma-mutability rather than UV mutability.     

Transfectability of rough strains of Salmonella typhimurium.

Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl2. The yield of infectious centers is about 10(-8) per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate-galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are not longer competent for transfection.