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.     

Analysis of pSC138, the multidrug resistance plasmid of Salmonella enterica serotype Choleraesuis SC-B67

Salmonella enterica serotype Choleraesuis (S. Choleraesuis) usually causes systemic infections in man and needs antimicrobial treatment. Multidrug resistance (MDR) in S. Choleraesuis is thus a great concern in the treatment of systemic non-typhoid salmonellosis. A large plasmid, pSC138, was identified in 2002 from a S. Choleraesuis strain SC-B67 that was resistant to all antimicrobial agents commonly used to treat salmonellosis, including ciprofloxacin and ceftriaxone. Complete DNA sequence of the plasmid had been determined previously (Chiu et al., 2005). In the present study, the sequence of pSC138 was reannotated in detail and compared with several newly sequenced plasmids. Some transposable elements and drug resistance genes were further delineated. Plasmid pSC138 was 138,742 bp in length and consisted of 177 open reading frames (ORFs). While 134 of the ORFs displayed significant identity levels to other plasmid and prokaryotic sequences, the remaining 43 ORFs have not been previously reported. Mobile elements, including two integrons, seven insertion sequences and eight transposons, and a truncated prophage together encompass at least 66,781 bp (48.1%) of the plasmid genome. The sequence of pSC138 consists of three major regions: a large composite transposable region Tn6088 with a Tn21-like backbone inserted by a variety of integrons or transposable elements; a transfer/maintenance region that contains a conserved ISEcp1-mediated transposon-like element Tn6092, carrying an AmpC gene, bla(CMY-2), that confers the ceftriaxone resistance; and a Rep_3 type of replication region. Another seven bacteremic strains of S. Choleraesuis that expressed the same MDR phenotype were identified during 2003-2008. The same Rep_3 type replicase and the bla(CMY-2)-containing, ISEcp1-mediated transposon-like element were found in the MDR isolates, suggesting a successful preservation and dissemination of the MDR plasmid. Comparison of pSC138 with other recently published plasmids revealed a high identity level between partial sequences of pSC138 and plasmids of the same or different incompatibility groups. The large MDR region found in pSC138 may provide a niche for the future evolution of the plasmid by acquisition of relevant resistance genes through the panoply of mobile elements and illegitimate recombination events.     

Regulation of spvR gene expression of Salmonella virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis

The expression regulation of spvR, a regulatory gene on the virulence plasmid (pKDSC50) of Salmonella choleraesuis serovar Choleraesuis, was investigated by spvR–lacZ translational fusion. The spvR gene was found to be positively regulated by its own product, the SpvR protein, and this unusual positive auto-regulation was repressed by the products of spvA and spvB, virulence-associated genes present downstream from the spvR gene. Amino acid sequence analysis revealed that the amino-terminal region of SpvB had homology with the CatM repressor protein of Acineto-bacter calcoaceticus, which belongs to the MetR/LysR protein family. On the other hand, the sigma factor RpoS was required for expression of the spvR gene in the stationary phase of bacterial growth. The SpvR protein was also necessary for self-activation, suggesting that an RNA polymerase holoenzyme containing RpoS requires SpvR protein in order to recognize the spvR promoter.     

Bile-induced curing of the virulence plasmid in Salmonella enterica serovar Typhimurium

Exposure to bile induces curing of the virulence plasmid in Salmonella enterica serovar Typhimurium (pSLT). Disruption of the ccdB gene increases pSLT curing, both spontaneous and induced by bile, suggesting that the pSLT ccdAB genes may encode a homolog of the CcdAB addiction module previously described in the F sex factor. Unlike the F sex factor, synthesis of pSLT-encoded pili does not confer bile sensitivity. These observations may provide insights into the evolution of virulence plasmids in Salmonella subspecies I, as well as the causes of virulence plasmid loss in other Salmonella subspecies.     

Complete genome sequence of Salmonella enterica serovar pullorum RKS5078

Salmonella enterica serovar Pullorum is a chicken-adapted pathogen, causing pullorum disease. Its strict host adaptation has been suspected to result in gene decay. To validate this hypothesis and identify the decayed genes, we sequenced the complete genome of S. Pullorum RKS5078. We found 263 pseudogenes in this strain and conducted functional analyses of the decayed genes.     

Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis

The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function.     

Complete genome sequence of Salmonella enterica subsp. enterica serovar Typhi P-stx-12

We report here the complete genome sequence of Salmonella enterica subsp. enterica serovar Typhi P-stx-12, a clinical isolate obtained from a typhoid carrier in India.     

The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen

Salmonella enterica serovar Choleraesuis (S.Choleraesuis), a highly invasive serovar among non-typhoidal Salmonella, usually causes sepsis or extra-intestinal focal infections in humans. S.Choleraesuis infections have now become particularly difficult to treat because of the emergence of resistance to multiple antimicrobial agents. The 4.7 Mb genome sequence of a multidrug-resistant S.Choleraesuis strain SC-B67 was determined. Genome wide comparison of three sequenced Salmonella genomes revealed that more deletion events occurred in S.Choleraesuis SC-B67 and S.Typhi CT18 relative to S.Typhimurium LT2. S.Choleraesuis has 151 pseudogenes, which, among the three Salmonella genomes, include the highest percentage of pseudogenes arising from the genes involved in bacterial chemotaxis signal-transduction pathways. Mutations in these genes may increase smooth swimming of the bacteria, potentially allowing more effective interactions with and invasion of host cells to occur. A key regulatory gene of TetR/AcrR family, acrR, was inactivated through the introduction of an internal stop codon resulting in overexpression of AcrAB that appears to be associated with ciprofloxacin resistance. While lateral gene transfer providing basic functions to allow niche expansion in the host and environment is maintained during the evolution of different serovars of Salmonella, genes providing little overall selective benefit may be lost rapidly. Our findings suggest that the formation of pseudogenes may provide a simple evolutionary pathway that complements gene acquisition to enhance virulence and antimicrobial resistance in S.Choleraesuis.     

Complete genomic sequence of Salmonella enterica serovar Enteritidis phage SE2

Salmonella enterica serovar Enteritidis has remained a major food-borne pathogen in humans. We isolated a virulent S. enterica serovar Enteritidis bacteriophage, SE2, which belongs to the family Siphoviridae. Phage SE2 could lyse S. enterica serovar Enteritidis PT-4, and its virulence was maintained even at ambient temperature. The genomic sequence of phage SE2 was composed of 43,221 bp with close similarity to those of Salmonella phage SETP3 and Salmonella phage SS3e. The strong and stable lytic activity of this phage might enable its use as a therapeutic or biocontrol agent against S. enterica serovar Enteritidis.     

Complete genome sequence of Salmonella enterica serovar typhimurium bacteriophage SPN1S

To understand the interaction between the host of pathogenic Salmonella enterica serovar Typhimurium and its bacteriophage, we isolated the bacteriophage SPN1S. It is a lysogenic phage in the Podoviridae family and uses the O-antigen of lipopolysaccharides (LPS) as a host receptor. Comparative genomic analysis of phage SPN1S and the S. enterica serovar Anatum-specific phage ε15 revealed different host specificities, probably due to the low homology of host specificity-related genes. Here we report the complete circular genome sequence of S. Typhimurium-specific bacteriophage SPN1S and show the results of our analysis.     

Complete genome sequence of Salmonella enterica serovar Typhimurium bacteriophage SPN3UB

Salmonella is one of the major pathogenic bacteria that cause food poisoning. To elucidate the host infection mechanism of Salmonella enterica serovar Typhimurium-targeting phages, the bacteriophage SPN3UB was isolated from a chicken fecal sample. This phage belongs morphologically to the Siphoviridae family and infects the host via the O antigen of lipopolysaccharide (LPS). To further understand its infection mechanism, we completely sequenced and analyzed the genome. Here, we announce its complete genome sequence and report major findings from the genomic analysis results.     

Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.     

Macrophage apoptosis associated with Salmonella enterica serovar Typhi plasmid

The present study was undertaken to investigate the relationship between plasmid isolated from S. enterica serovar Typhi (pR(ST98)) and macrophage apoptosis. pR(ST98) was transferred into an attenuated S. enterica serovar Typhimurium strain RIA to create a transconjugant pRsT98/RIA. Standard S. enterica serovar Typhimurium virulence strain SR-11 was used as a positive control, and RIA as a negative one. Murine macrophage-like cell line (J774A.1) was used as an infectious cell model in vitro. In order to determine the inhibition and bactericidal effect of amikacin (AMK) to extracellular bacteria and the best optimization co-culture ratio between Salmonella and J774A.1, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AMK to strains SR-11, pR(ST98)/RIA and RIA and multiplicity of infection (MOI) were detected first, and then J774A.1 was infected by the above three serovar Typhimurium strains. Apoptosis of J774A.1 was examined with electron microscopy and flow cytometry after annexin-V/propidium iodide labeling at 0, 1, 3, 6, 12 and 24 h. Mitochondrial membrane potential was detected by JC-1 staining method. It was demonstrated that MIC of AMK to the three strains was 10 microg/ml, MBC was 80 microg/ml, and optimal MOI was 100:1. pR(ST98)/RIA resulted in a higher apoptosis of J774A.1 than RIA, apoptotic features such as chromatin margination could be observed after 3 h, and death of J774A.1 cells was associated with the loss of mitochondrial membrane potential. These results indicated that pR(ST98) could enhance the virulence of its host bacteria, evidenced by increased macrophage apoptosis.     

Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis.     

Polyamines are required for virulence in Salmonella enterica serovar Typhimurium

Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection, indicating that these polyamines function as an environmental signal that primes S. Typhimurium for intracellular survival. Accordingly, experiments addressed at elucidating the roles of these polyamines in infection revealed that expression of genes from both of the major virulence loci SPI1 and SPI2 responded to exogenous polyamines and was reduced in the polyamine mutant. Together our data demonstrate that putrescine and spermidine play a critical role in controlling virulence in S. Typhimurium most likely through stimulation of expression of essential virulence loci. Moreover, our data implicate these polyamines as key signals in S. Typhimurium virulence.     

Genetic map of the virulence plasmid of Salmonella enteritidis and nucleotide sequence of its replicons

Partial sequencing of a genomic library of the virulence plasmid of Salmonella enteritidis has been used to localize in the restriction map of this plasmid the genetic loci already described in other Salmonella plasmids. The comparison of the vestigial tra region with the corresponding genes in the F plasmid allowed us to define the extent of the deletions that the S. enteritidis plasmid should have suffered. The putative replicons of the plasmid, repB and repC, were isolated and both proved to be functional in Escherichia coli, but repC was segregationally unstable. The nucleotide sequence of repB showed the typical organization of RepFIIA replicons, although the similarity was lower than usual in this group of replicons. The highest homology was found with the replicon of the virulence plasmid pYVe439-80 from Yersinia enterocolitica (72.5%). Replicon repC also showed a maximum identity of 72.6% with known replicons, namely the RepFIB of pColV-K30 and P307, both virulence plasmids isolated from E. coli. We conclude that the S. enteritidis plasmid could arise from the S. typhimurium plasmid through deletions, and that they are evolutionary distant from other IncFI and IncFII plasmids.