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Epidemiological studies on Salmonella in a certain area ("Walcheren project"). I. The presence of Salmonella in man, pigs, insects, seagulls and in foods and effluents.

During a certain period various materials (pigs, foods, insects, seagull droppings, chopping-block scrapings from butcher's shops, effluents of sewage treatment plants and stools of patients) were examined for the presence of Salmonella at the same time in a relatively small area (Walcheren). Certain types of Salmonella (S. typhi murium type II 505, S. panama, S. infantis and S. brandenburg) were frequently isolated from almost all materials examined. This may indicate the existence of Salmonella contamination cycles: one may think of the cycle: slaughter animal (infected from the environment and/or by meal) - meat - consumer - patient or healthy carrier - effluent and surface water - insects, birds and rodents - slaughter animal or meat and possibly other foods - consumer.

Animals↗

[Report on Salmonella isolates submitted to the German National Veterinary Salmonella Reference Laboratory in the year 1999].

The National Salmonella Reference Laboratory (NRL-Salm) of the Federal Institute for Health Protection of Consumers and Veterinary Medicine receives putative Salmonella isolates originating from animals, food, feed and the environment for typing. This report summarises the results of the sero- and phagetyping studies. In livestock S. Typhimurium DT 104 with 24% and S. Enteritidis PT 4 with 9% of all isolations predominate like in the human isolates. These sero- and phagetypes are frequently isolated from food as well and consequently can reach the consumer via the food chain.

Animals↗

The effect of the Salmonella genomic island 1 on in vitro global gene expression in Salmonella enterica serovar Typhimurium LT2.

A Salmonella genomic island 1 (SGI1) isogenic strain pair was constructed using Salmonella enterica serovar Typhimurium LT2 (ST LT2). Real-time quantitative reverse transcriptase PCR revealed detectable mRNA transcripts for all 44 putative ORFs encoded within the SGI1. The highest levels of transcripts observed in SGI1 encoded ORFs were found in genes conferring antibiotic resistance to ampicillin, streptomycin/spectinomycin, and sulphonamides. Abundant mRNA transcripts, relative to gapA, were also noted for one putative regulatory ORF and seven ORFs of unknown function encoded within SGI1, whose products could represent factors contributing to increases in virulence and/or fitness of the organism. DNA microarray analysis revealed the differential expression of known factors that contribute to virulence in many pathogens. Twenty-two chromosomal genes were significantly upregulated in ST LT2 harboring SGI1, which included increased expression of iron and sialic acid utilization genes. Decreased expression was noted for 15 genes in ST LT2 harboring SGI1, including genes involved in chemotaxis and motility. This is the first report examining gene expression within the SGI1, as well as its potential effect on global gene expression, and sets the foundation for future studies involving the effect of SGI1 in other Salmonella spp.

Conjugation, Genetic↗

Salmonella fimbriae: novel antigens in the detection and control of salmonella infections.

Fimbriae are thin, proteinaceous surface organelles produced by members of the Enterobacteriaceae, including most salmonellas. A number of fimbrial antigens expressed by strains of Salmonella enteritidis and S. typhimurium have now been described and characterized. However, their functions are still poorly understood, although some evidence indicates they have a role in bacterial survival in the host or external environment. Diagnostic tests based on the detection of fimbriae or specific antibodies against them have recently been developed and applied successfully to the rapid and specific identification of S. enteritidis infections. The role of salmonella fimbriae in future generations of live vaccines either as protective antigens or as the carriers of heterologous antigens is also discussed.

Bacterial Vaccines↗

The correlation between serum IgA antibody levels and resistance to infection with Salmonella typhimurium after oral immunization with various salmonellae.

Subsequent to oral feeding of various Salmonella strains to mice, the IgA antibodies directed against the O-somatic antigens of the immunising strain were measured in both serum and intestinal juice, using the ELISA technique. 21 days after oral immunization the IgA antibody levels were relatively high only in those mice which were resistant to challenge with a virulent strain of Salmonella typhimurium. Because there was no correlation between protection and the specificity of the 'O' somatic antigens of the immunising and challenge strains, the IgA antibodies measured were not responsible for protection. Nevertheless, the level of these antibodies affords a good index of the resistance of mice or immune status of mice to Salmonella infection.

Administration, Oral↗

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group U (o 43) lipopolysaccharides.

1. A series of oligosaccharides was isolated from Salmonella milwaukee lipopolysaccharide by partial acid hydrolysis. 2. Structural studies on these oligosaccharides indicated that the O-specific side chain of this lipopolysaccharide has a repeating pentasaccharide unit that is probably alpha-d-galactosyl-(1-->3)-beta-d-galactosyl- (1-->3)-N-acetylgalactosaminyl-(1-->3)-N-acetyl- d-glucosaminyl-(1-->4)-l-fucose. 3. Another oligosaccharide, which is not structurally related to the repeating pentasaccharide unit, has also been isolated and it is indistinguishable from an oligosaccharide obtained from Salmonella ;rough' (R) lipopolysaccharides. The isolation of this and similar core oligosaccharides from all chemotype VI lipopolysaccharides supports the view that Salmonella S-lipopolysaccharides have a common core that is probably identical with RII lipopolysaccharide.

Antigens↗

Identification of a novel core type in Salmonella lipopolysaccharide. Complete structural analysis of the core region of the lipopolysaccharide from Salmonella enterica sv. Arizonae O62.

For the first time, the complete structure of a lipopolysaccharide (LPS) core region from Salmonella enterica has been identified that is different from the Ra core type generally thought to be present in all Salmonella LPS. The LPSs from two rough mutants and the smooth form of S. enterica sv. Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy. In the novel core type, the terminal D-GlcNAc residue present in the Ra core type, is replaced by a D-Glc residue. The O-specific polysaccharide is alpha1-->4-linked to the second distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 of L-glycero-D-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II). [structure: see text] Abbreviations in Structure I are as follows: Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide. The presence of this novel core type in LPS of S. enterica should be taken into account in the development of a general antibody-based diagnostic system for Salmonella.

Carbohydrate Conformation↗

Oral immunization with live, avirulent fla+ strains of Salmonella protects mice against subsequent oral challenge with Salmonella typhimurium.

Some strains of Salmonella, when fed to mice, establish a nonlethal, limited infection in the Peyer's patches of the small intestine. When such mice are later challenged orally with a normally lethal dose of virulent Salmonella typhimurium, a protective effect of the prior vaccination is seen. In an effort to characterize the determinant(s) of the avirulent strains responsible for this protective effect, we examined the cell envelope protein profiles of five such protective strains and of eight strains of Salmonella that were both nonpersistent and nonprotective when fed to mice. The protective strains produced high levels of flagellin. We made otherwise isogenic fla+ and fla- derivatives of two such strains and showed that although the fla- derivatives colonized mice as well as did the fla+ strains, the fla- derivatives given orally showed a much reduced ability to protect mice from S. typhimurium challenge. Both fla+ and fla- strains induced cellular immunity in vaccinated mice.

Administration, Oral↗

Class 1 integrons in various Salmonella enterica serovars isolated from animals and identification of genomic island SGI1 in Salmonella enterica var. Meleagridis.

OBJECTIVES: To determine the prevalence of integron-mediated antibiotic resistance in a diverse sample set of Salmonella enterica isolated from animals. MATERIALS AND METHODS: Multiplex PCR was used to detect class 1 integron gene sequences, and integron gene cassettes were identified by PCR mapping. Susceptibility to 18 antibiotics or antibiotic combinations commonly used in either human or veterinary medicine was measured using a microdilution method, and statistical comparisons of the frequency of resistance between groups were made using Fisher's two-sided probability test. Genotypic comparisons of isolates were made following pulsed-field gel electrophoresis of genomic DNA. RESULTS: Thirty-two (30.8%) of 104 isolates contained class 1 integron sequences. Integron-positive isolates represented 15 different S. enterica serovars, were obtained from nine different animal species and had a higher frequency of non-integron-mediated antibiotic resistance (P < 0.05) compared with integron-negative isolates. One non-Typhimurium isolate (S. enterica Meleagridis) contained an SGI1 genomic island, including the antibiotic resistance gene cluster. CONCLUSIONS: These data demonstrate that integron-mediated antibiotic resistance is common among diverse Salmonella serovars, many of them rare. In addition, SGI1 is not limited to Salmonella enterica Typhimurium DT104 or other commonly isolated serovars.

Animals↗

Isolation of Salmonella enterica subspecies enterica serovar Paratyphi B dT+, or Salmonella Java, from Indonesia and alteration of the d-tartrate fermentation phenotype by disrupting the ORF STM 3356.

Salmonella enterica subspecies enterica serovar Paratyphi B [O1,4,(5),12 : Hb : 1,2] can cause either an enteric fever (paratyphoid fever) or self-limiting gastroenteritis in humans. The d-tartrate non-fermenting variant S. enterica subsp. enterica serovar Paratyphi B dT- (S. Paratyphi B) is the causative agent of paratyphoid fever, and the d-tartrate fermenting variant S. enterica subsp. enterica serovar Paratyphi B dT+ (S. Paratyphi B dT+; formerly called Salmonella Java) causes gastroenteritis. S. Java is currently recognized as an emerging problem worldwide. Twelve dT+ S. Java isolates were collected in Indonesia between 2000 and 2002. One-third of them contained Salmonella genomic island 1 (SGI1), which gives the multidrug-resistant phenotype to the bacteria. In this study, a PCR-based method to detect a single nucleotide difference responsible for the inability to ferment d-tartrate, reported elsewhere, was validated. The d-tartrate fermenting phenotype of S. Java was converted to the non-fermenting phenotype by the disruption of the ORF STM 3356, and the d-tartrate non-fermenting phenotype of the ORF STM 3356-disrupted strain and the dT- reference strain was changed to the dT+ phenotype by complementing ORF STM 3356 in trans. The results show that the dT+ phenotype requires a functional product encoded by STM 3356, and support the use of the PCR-based discrimination method for S. Paratyphi B and S. Java as the standard differentiation method.

Anti-Bacterial Agents↗

Glyceraldehyde phosphate at the reducing terminus of Salmonella Q haptens. Salmonella montevideo.

The O antigen polysaccharide of Salmonella montevideo was isolated from a core-defective mutant by the phenol/water procedure, and was suspected to contain phosphomonester and cyclic phosphodiester at its reducing end in anology to the O hapten from Salmonella typhimurium (Kent and Obsborn, 1968. Therefore, it was chromatographed on a DEAE-cellulose column. Whereas one part eluted with water the other part of the polysaccharide could only be eluted with buffer. Both fractions were further purified on Sephadex G100 and contained mannose, glucose, N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: less than 0.1. In order to specifically label the reducing end phosphate was removed enzymatically, or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions were reduced with sodium horo[3H]hydride. Both fractions yield mainly [3H]glycerol after hydrolysis and paper chromatogaphy. In addition, [3H]mannitol and [H]monohydroxyacetone could be identified by paper chromatography and were concluded to be the result of phosphate migration and beta-elimination reactions taking place during the isolation procedure and the various treatments prior to sodium boro[3H]hydride reduction. These findings in addition to periodate oxidation studies indicated that the O antigen polysaccharide of Salmonella montevideo had glyceraldehyde phosphate at its reducing end. From the incorporation of 3H into the polysaccharide the O antigen was calculated to consist of about 19 repeating units of 6 sugar residues each.

Acid Phosphatase↗

Geographically dependent distribution of gyrA gene mutations at codons 83 and 87 in Salmonella Hadar, and a novel codon 81 Gly to His mutation in Salmonella Enteritidis.

In all, 90 nalidixic acid-resistant clinical strains of Salmonella Hadar and Salmonella Enteritidis isolated in Norway but of predominantly foreign origin were subjected to sequencing of the gyrA, gyrB, parC and parE genes. All the isolates contained at least one mutation in gyrA codon 83 or codon 87. A highly significant correlation between mutations in gyrA codon 83 and strains originating from Southeast Asia was found in S. Hadar but not in S. Enteritidis. A novel gyrA codon 81 Gly to His mutation was discovered in one S. Enteritidis isolate. One amino-acid (aa) changing mutation was found outside the quinolone resistance-determining region (QRDR) of S. Hadar parC at codon 57, which has previously only been observed once in Salmonellae.

Codon↗

Multiple-antibiotic resistance in Salmonella enterica serotype Paratyphi B isolates collected in France between 2000 and 2003 is due mainly to strains harboring Salmonella genomic islands 1, 1-B, and 1-C.

This study was conducted to investigate the occurrence of multiple-antibiotic resistance among 261 clinical isolates of Salmonella enterica serotype Paratyphi B strains collected between 2000 and 2003 through the network of the French National Reference Center for Salmonella. The 47 multidrug-resistant (MDR) isolates identified (18%), were characterized on the basis of the presence of several resistance genes (bla(TEM), bla(PSE-1), bla(CTX-M), floR, aadA2, qacEdelta1, and sul1), the presence of Salmonella genomic island 1 (SGI1) by PCR mapping and hybridization, and the clonality of these isolates by several molecular (ribotyping, IS200 profiling, and pulsed-field gel electrophoresis [PFGE]) and phage typing methods. The results of PCR and Southern blot experiments indicated that 39 (83%) of the 47 S. enterica serotype Paratyphi B biotype Java MDR isolates possessed the SGI1 cluster (MDR/SGI1). Among these 39 MDR/SGI1 isolates, only 3 contained variations in SGI1, SGI1-B (n = 1) and SGI1-C (n = 2). The 39 MDR/SGI1 isolates showed the same specific PstI-IS200 profile 1, which contained seven copies from 2.6 to 18 kb. Two PstI ribotypes were found in MDR/SGI1 isolates, RP1 (n = 38) and RP6 (n = 1). Ribotype RP1 was also found in two susceptible strains. Analysis by PFGE using XbaI revealed that all the MDR/SGI1 isolates were grouped in two related clusters, with a similarity percentage of 82%. Isolation of MDR/SGI1 isolates in France was observed mainly between the second quarter of 2001 and the end of 2002. The source of the contamination has not been identified to date. A single isolate possessing the extended-spectrum beta-lactamase bla(CTX-M-15) gene was also identified during the study.

Adolescent↗

Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments.

Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimurium porin-deficient mutants. In this study, isogenic transposon mutants of S. typhimurium with insertions in ompR, envZ, ompF, ompC, ompD, osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that ompR and envZ mutants were reduced or completely inhibited for the formation of Salmonella-induced filaments (Sifs). This result was confirmed with an ompB deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella. Genetic analysis showed that the ompR mutation could be complemented in trans by cloned ompR to restore its ability to induce Sifs. In contrast, mutations in the known ompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S. typhimurium.

Bacterial Outer Membrane Proteins↗

Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype typhimurium.

The enteric pathogen Salmonella enterica serotype Typhimurium induces apoptosis in infected macrophages. This process is rapid, specific, and depends on the type III protein secretion system encoded within Salmonella pathogenicity island 1 (SPI1). Here, we demonstrate that serotype Typhimurium can activate programmed macrophage cell death independently of SPI1. SPI1 independent induction of apoptosis in infected macrophages is observed as early as 12 to 13 h postinfection, even in the absence of intracellular bacterial replication. Delayed activation of programmed macrophage cell death is not observed with serotype Typhimurium strains mutated in ompR or SPI2. Even though SPI2 mutants have a defect in intracellular proliferation, our results indicate that long-term intracellular survival and growth are not required for delayed macrophage killing per se, since Salmonella mutants that are severely defective in intracellular growth still induce delayed apoptosis. Inactivation of genes required for either rapid or delayed induction of apoptosis results in a conditional noncytotoxic phenotype, whereas simultaneous inactivation of genes required for both rapid and delayed induction of apoptosis renders serotype Typhimurium noncytotoxic under all conditions tested. Our hypothesis is that differential activation of programmed macrophage cell death by serotype Typhimurium occurs under discrete physiological conditions at distinct locations within an infected host.

Animals↗

HLA-B27-transfected (Salmonella permissive) and HLA-A2-transfected (Salmonella nonpermissive) human monocytic U937 cells differ in their production of cytokines.

The cytokine secretion of the Salmonella-permissive, HLA-B27-positive U937 cells was examined, as it was previously shown that these cells kill Salmonella less efficiently than controls. Salmonella-permissive U937 cells showed upregulated production of interleukin 10 and to a lesser extent tumor necrosis factor alpha. HLA-B27-associated modulation of cytokine responses may have importance in the pathogenesis of reactive arthritis.

Arthritis, Reactive↗

Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar typhimurium in the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the Salmonella-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this Salmonella pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of sifA mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells.

Antigens, CD↗

Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella virginia O:8.

The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure: [formula; see text] The disaccharide structure alpha-D-Abep-(1----3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of alpha-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum.

Carbohydrate Conformation↗