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.     

Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.     

Rapid identification of Salmonella enterica serovars, Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum, by multiplex PCR

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.     

Reduced amounts of LPS affect both stress tolerance and virulence of Salmonella enterica serovar Dublin

Signature-tagged mutagenesis (STM) is a widely used technique for identification of virulence genes in bacterial pathogens. While this approach often generates a large number of mutants with a potential reduction in virulence a major task is subsequently to determine the mechanism by which the mutations influence virulence. Presently, we have characterised a Salmonella enterica serovar Dublin STM mutant that, in addition to having reduced virulence, was also impaired when growing under various stress conditions. The mutation mapped to the manC (rfbM) gene of the O-antigen gene cluster involved in O-antigen synthesis. The O-antigen is a component of the lipopolysaccharide (LPS) forming a unique constituent of the outer membrane of Gram-negative bacteria. While mutations in the O-antigen genes usually eliminate the entire O-antigen side chain we found that the transposon mutant produced intact O-antigen, however, the mutation reduced the amount of LPS.     

Precise excision and secondary transposition of TnphoA in non-motile mutants of a Salmonella enterica serovar Enteritidis clinical isolate

Mutagenesis with TnphoA has been widely used in many bacteria. Here, we report the excision and secondary transposition of this transposon in three non-motile (fliC, fliF and motB) mutants of Salmonella enterica serovar Enteritidis (S. Enteritidis). Isolation of motile revertants showed that they were kanamycin resistant and conserved a copy of TnphoA in their genome in an insertion site different from the initial one. They also expressed an intact flagella. Characterization of the motile revertant derived from the fliC mutant showed that TnphoA excised precisely from the fliC gene, resulting in an equivalent amount of FliC secreted protein in the revertant compared to that of the wild-type strain. These results show that TnphoA mutants should be used with care and underline the value of using transposon derivatives lacking the transposase gene.     

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis,Dublin and Gallinarum

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.     

Inactivation of the flagellin gene of Salmonella enterica serotype enteritidis strongly reduces invasion into differentiated Caco-2 cells

A nonflagellated mutant of Salmonella enterica serotype Enteritidis was constructed by disrupting the flagellin gene (fliC). Northern blot analysis indicated that the mutation did not affect expression of the downstream fliU gene. Infection experiments with differentiated Caco-2 cells revealed that the mutant was about 50-fold less invasive than the wild-type strain, while bacterial adherence was unaffected. Complementation of the mutant with an intact fliC copy restored flagella formation and efficient bacterial invasion. Our data demonstrate that the fliC gene of S. enterica serotype Enteritidis is essential for the invasion of Caco-2 cells.     

Functional and molecular characterization of pSE34 encoding a type IV secretion system in Salmonella enterica serotype Enteritidis phage type 34

Salmonella enterica serotype Enteritidis infection remains a serious public health threat to humans. Salmonella Enteritidis phage type 4 (PT4) is a clone that has already caused a global pandemic for years. To investigate why PT34 becomes a subdominantly emerging phage type, molecular characterizations, including serotyping, pulsed-field gel electrophoresis (PFGE), phage typing, and plasmid profiling, were carried out on PT34. The results indicated that relative to PT4, PT34 contained an additional 32-kb DNA segment in PFGE and a 33-kb plasmid pSE34 in plasmid profiling. Southern blot hybridization showed that the DNA segment was the major part of pSE34. All of the S . Enteritidis PT34 clinical isolates possessed pSE34, while PT4 and PT21 did not. Sequencing analysis revealed that pSE34 is 32 950 bp long, with a G+C% content of 41.2%, and contains a total of 53 orf s. Transposon mutagenesis demonstrated that taxB, taxC , and the pilX operon on this plasmid participated in the process of conjugation. In virulence testing, PT34 that harbored pSE34, compared with PT4, showed no increased invasion to tissue culture cells in vitro . The presence of conjugative pSE34 in PT4 caused the conversion of phage type from PT4 to PT34, suggesting that the emergence of PT34 was a result of the introduction of the conjugative pSE34 into its common progenitor PT4.     

The use of a bacteriophage cocktail as a biocontrol measure to reduce Salmonella enterica serovar Enteritidis contamination in ground meat and goat cheese

We evaluated the effectiveness of phages on meats and goat cheese contaminated with Salmonella Enteritidis (SE). In meats, reductions of SE were observed during the whole experiment, while in goat cheese a reduction was only observed at day 3. We discuss the relevance of phages as a biocontrol in food.     

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.     

High concentration of sodium chloride could induce the viable and culturable states of Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis

In the present study, Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were transferred into Luria–Bertani medium without NaCl (LBWS) and adjusted to various pHs (4, 5, 6 and 7) with lactic acid containing 0·75, 5, 10 and 30% NaCl, and stored at 25°C until the bacterial populations reached below detectable levels on tryptic soy agar (TSA). Although E. coli O157:H7 and S. Enteritidis did not grow on TSA when incubated in LBWS with 30% NaCl for 35 and 7 days, more than 60 and 70% of the bacterial cells were shown to be viable via fluorescent staining with SYTO9 and propidium iodide (PI), respectively, suggesting that a number of cells could be induced into the viable but nonculturable (VBNC) state. These bacteria that were induced into a VBNC state were transferred to a newly prepared tryptic soy broth (TSB) and then incubated at 37°C for several days. After more than 7 days, E. coli O157:H7 and S. Enteritidis regained their culturability. We, therefore, suggest that E. coli O157:H7 and S. Enteritidis entered the VBNC state under the adverse condition of higher salt concentrations and were revived when these conditions were reversed.     

Epidemiological analysis of Salmonella enterica ssp. enterica serovars Hadar, Brancaster and Enteritidis from humans and broiler chickens in Senegal using pulsed-field gel electrophoresis and antibiotic susceptibility

AIMS: Salmonella Hadar, Salmonella Brancaster and Salmonella Enteritidis are the main Salmonella enterica ssp. enterica serovars isolated from poultry in Senegal. Our objective was to analyse the pulsed-field gel electrophoresis (PFGE) and antibioresistance patterns of strains belonging to these serovars and to assess the significance of broiler-chicken meat as a source of human infection. METHODS AND RESULTS: A total of 142 Salmonella isolates were analysed: 79 were isolated from Senegalese patients with sporadic diarrhoea (11 S. Hadar, nine S. Brancaster and 59 S. Enteritidis) and 63 from poultry (30 S. Hadar, 17 S. Brancaster and 16 S. Enteritidis). The PFGE of XbaI- and SpeI-digested chromosomal DNA gave 20 distinct profiles for S. Hadar, nine for S. Brancaster and 22 for S. Enteritidis. Each serovar was characterized by a major pulsotype which was X3S1 in 42% of S. Hadar, X8S1 in 53.8% of S. Brancaster and X1S2 in 43% of S. Enteritidis isolates. Human and poultry isolates of Salmonella had common PFGE patterns. Antibiosensitivity tests showed multiresistance (more than two drugs) was encountered in 14.5% of S. Hadar and in 5% of S. Enteritidis isolates. Resistance to quinolones was considered to be of particular importance and 14.5% of S. Hadar isolates were found to be resistant to nalidixic acid. CONLCUSIONS: The sharing of similar PFGE profiles among isolates from humans and poultry provided indirect evidence of Salmonella transmission from contaminated broiler meat. But most of the Salmonella isolates remained drug sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Efforts are needed to eliminate Salmonella from poultry meat intended for human consumption. This study has also highlighted the importance of continuous surveillance to monitor antimicrobial resistance in bacteria associated with animals and humans.     

Comparison of 40 Salmonella enterica serovars injured by thermal, high-pressure and irradiation stress

AIM: To investigate and compare the inherent resistance of 40 Salmonella serovars to heat, irradiation and high-pressure stress. METHODS AND RESULTS: D10 values for each of the three stresses were calculated for four serovars, chosen as representatives from a catalogue of 40. Based on these results, conditions for each stress were defined, which produced, on average, a three-log reduction in viability. Heat stress (57 degrees C for 13 min), high-pressure stress (350 MPa for 10 min at 20 degrees C) and irradiation stress (1.5 kGy at 20 degrees C) were applied to all 40 serovars in the collection. Injury and loss of viability for all serovars were determined. CONCLUSIONS: Cluster analysis identified five groupings of isolates in terms of resistance to the applied stresses. The independent response of each isolate to all three stresses suggests that there is no relationship between resistances. SIGNIFICANCE AND IMPACT OF THE STUDY: Each serovar is inherently different. For modelling of real-life food preservation processing the most resistant isolates for that process should be chosen. The results also emphasize the importance of including multiple stress resistant strains when food preservation systems apply multiple stresses.     

The presence of major world-wide clones for phage type 4 and 8 Salmonella enterica serovar Enteritidis and the evaluation of their virulence levels by invasiveness assays in vitro and in vivo

Seventy-seven animal isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from the United States were analyzed by phage typing and pulsed field gel electrophoresis (PFGE). Thirty-nine strains were found with phage types (PT) 4, 8, and 13a. When the chromosomal DNA of these 39 isolated strains with PT4, 8, and 13a were digested with XbaI, SpeI and NotI, followed by PFGE analysis, 28 strains were found with a pattern combination of X4S4N4, which was the major subtype. When PFGE patterns of the US isolates with PT 4 and 8 were compared with those of the Taiwanese and German isolates, pattern X3S3N3 was confirmed to be the world-wide subtype shared by PT 4 isolates, as previously reported, while pattern X4S4N4 was newly found to be the most common subtype shared by PT 8 strains. The presence of such major world-wide clones, however, does not necessarily mean that these clones are highly virulent, at least not according to the results of invasiveness assays using cultured human intestinal epithelium cell line Int-407 and living BALB/mice.     

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.     

Difference and consensus of whole cell Salmonella enterica subsp. enterica serovars matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra

AIM: Application of MALDI-TOF MS for characterization of strains of Salmonella enterica subsp. enterica. METHODS AND RESULTS: Whole cells were analysed by MALDI-TOF MS. Spectra with a maximum of 500 mass peaks between (m/z) 0 and 25000 were examined for consensus peaks manually and by a computer software algorithm. Consensus peaks were observed by both methods for spectra of Salmonella enterica serovars Derby, Hadar, Virchow, Anatum, Typhimurium and Enteritidis. CONCLUSIONS: Differences in numbers of consensus peaks in spectra obtained by manual and computer comparison indicated that development of the software involving statistical analysis of peak accuracy is necessary. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of an analysis system for peak profiles in whole cell MALDI-TOF MS spectra to enable intra and interlaboratory comparison.