Salmonella virulence plasmid. Modular acquisition of the spv virulence region by an F-plasmid in Salmonella enterica subspecies I and insertion into the chromosome of subspecies II,IIIa, IV and VII isolates.

The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulence plasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulence plasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulence plasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae.     

Identification of a conjugative plasmid carrying antibiotic resistance and salmonella plasmid virulence (spv) genes in epidemic strains of Salmonella typhimurium phage type 193

A plasmid pDEP34 that codes for resistance to ampicillin, streptomycin, sulphonamides and tetracyclines has been identified in strains of Salmonella typhimurium phage type 193 which have become increasingly common in England and Wales since 1988. pDEP34 is also self-conjugative, carries the genes responsible for the virulence of host strains for BALB/c mice ( spv genes) and is closely related to the Salm. typhimurium 'serotype-specific' plasmid pSLT.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe

M.D. HAMPTON, E.J. THRELFALL, J.A. FROST, L.R. WARD AND B. ROWE. 1995. Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991–92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%). Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm. dublin serovar-specific plasmid. On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized. Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol. wt plasmid which coded for tetracycline resistance only. Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting. These results demonstrate that Salm. typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance

Among more than 2,500 serovars, eight contain a virulence plasmid, including medically important Salmonella enterica serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium. These serovar-specific virulence plasmids vary in size, but all contain the spv operon, which plays a role in the expression of the virulence. Genetically, these virulence plasmids are likely derived from a common ancestral plasmid possessing virulence-related genes and loci. Based on the analysis of the available DNA sequences of the plasmids, the phylogenetic path may be split into two: pSPV (virulence plasmid of S. Gallinarum-Pullorum) acquires an incompatibility-related locus that differs from that of the others. At some point, pSCV (virulence plasmid of S. Choleraesuis) and pSDV (virulence plasmid of S. Dublin) lose oriT by recombination or simply by deletion, making the two unable to be mobilized. On the other hand, pSEV (virulence plasmid of S. Enteritidis) also loses some DNA by deletion but not as extensively as pSCV, and therefore pSEV is closest to pSTV (virulence plasmid of S. Typhimurium) both genetically and biologically. The pSTV shows the least alternation during the evolution. There are two types of pSDV. pSDVu recombines with non-virulence 36.6-kb plasmid to acquire additional incompatibility trait to form pSDVr. Recent reports indicated that S. Choleraesuis and S. Typhimurium could generate different types of hybrid plasmids, which consisted of the serovar-specific virulence plasmid and an array of resistance gene cassettes. The recombination gives Salmonella a survival advantage in an unfavorable drug environment. The integration of resistance genes and additional replicons into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and poses a serious threat to public health.     

Evolution of genes on the Salmonella Virulence plasmid phylogeny revealed from sequencing of the virulence plasmids of S. enterica serotype Dublin and comparative analysis

Salmonella enterica serotype Dublin harbors an approximately 80-kb virulence plasmid (pSDV), which mediates systemic infection in cattle. There are two types of pSDV: one is pSDVu (pOU1113) in strain OU7025 and the other pSDVr (pOU1115) in OU7409 (SD Lane) and many clinical isolates. Sequence analysis showed that pSDVr was a recombinant plasmid (co-integrate) of pSDVu and a plasmid similar to a 35-kb indigenous plasmid (pOU1114) of S. Dublin. Most of the F-transfer region in pSDVu was replaced by a DNA segment from the pOU1114-like plasmid containing an extra replicon and a pilX operon encoding for a type IV secretion system to form pSDVr. We reconstructed the particular evolutionary history of the seven virulence plasmids of Salmonella by comparative sequence analysis. The whole evolutionary process might begin with two different F-like plasmids (IncFI and IncFII), which then incorporated the spv operon and fimbriae operon from the chromosome to form the primitive virulence plasmids. Subsequently, these plasmids descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. Our results suggest that the phylogeny of virulence plasmids as a result of frequent recombination provides the opportunity for rapid evolution of Salmonella in response to the environmental cues.     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

The influence of autophagy on mouse inflammatory responses caused by Salmonella enterica serovar Typhimurium with spv genes

An investigation into the effects of Salmonella plasmid virulence genes (spv) on autophagy, apoptosis, and inflammation was carried out in mice, using a strain of Salmonella enterica serovar Typhimurium (S. typhimurium) SR-11 carrying spv. Strain BRD509 without spv was used as a control. Results showed that the expression of autophagy protein Beclin-1 in the livers and spleens in the SR-11 group was lower than that in the BRD509 group, while the apoptosis protein, Caspase-3, was higher in the SR-11 group. Inflammatory cytokine levels [interleukin 12 (IL-12) and interferon γ (IFN-γ)] were higher in the SR-11 group compared with those in the BRD509 group since 4 d post-infection. In addition, we found an increase in severe pathological changes and larger viable bacterial amounts in livers and spleens in the SR-11 group. After intervention with autophagy agonist rapamycin (RAPA), Beclin-1 expression increased in both groups, while Caspase-3 expression was different between the two groups: Caspase-3 decreased in the SR-11 group but increased in the BRD509 group. Moreover, RAPA decreased cytokine levels, bacterial quantity and organ-related injury in the SR-11 group whereas RAPA increased cytokine levels and aggravated organ injury in the BRD509 group. Results from these studies suggest that S. typhimurium with spv genes may exacerbate infection by inhibiting autophagy and affecting the production of inflammatory cytokines. RAPA-enhanced autophagy may improve the secretion of cytokines in order to protect the host from damaging by Salmonella infection. Our study suggests that the regulation of cellular autophagy may play a role in the prevention and control of certain infectious diseases.     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

Abstract The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using proter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present with the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

Plasmid virulence gene expression induced by short-chain fatty acids in Salmonella dublin: identification of rpoS-dependent and rpo-S-independent mechanisms.

The Salmonella plasmid virulence spvABCD genes are growth phase regulated and require RpoS for maximal expression in stationary phase. We identified a growth phase-independent expression of spv which is mediated by short-chain fatty acids. During this fatty acid-mediated expression of spv, RpoS is required for induction only during exponential phase. In stationary phase, an rpoS-independent mechanism is responsible for expression of spv.     

Phage types and plasmid profiles of plasmid DNA strains of Salmonella enterica subsp. enterica ser. Enteritidis (S. Enteritidis) isolated from food poisoning outbreaks in 2001

Salmonella Enteritidis strains are the most often isolated Salmonella serovar in Poland. In the present study, phage typing, antibiotic resistance testing and plasmid profile analysis, have been applied to characterise 41 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 41 S. Enteritidis strains coming from Poland. All 41 strains were typable and 5 different phage types were observed. Among 41 strains tested, both PT6 and PT21 were recognized in the 15 strains (36.6%). Nine strains (22%) belonged to phage type 8. The others PTs were represented by small amount of strains (PT1var and PT4). Among all tested isolates only 4 different plasmid profiles were observed. Of the 41 strains investigated, 16 (39%) contained the 57 kb plasmid alone. The remaining 25 strains (61%) except 57 kb plasmid, possessed additional DNA particles. The probable phage type conversion of PT21 to PT1var strain, possibly connected with smaller DNA particle presence, was observed. This hypothesis needs confirmation. The real S. Enteritidis epidemiological situation in Poland should be known after introducing of systematic, annual research program.     

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.