Prevalence and Genetic Properties of Salmonella enterica Serovar Typhimurium Definitive Phage Type 104 Isolated from Rattus norvegicus and Rattus rattus House Rats in Yokohama City, Japan

Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.     

Characterization of the genomes of a diverse collection of Salmonella enterica serovar Typhimurium definitive phage type 104

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We completed the full DNA sequence of one DT104 strain, NCTC13348, and showed that significant differences between the genome of this isolate and the genome of the previously sequenced strain Salmonella serovar Typhimurium LT2 are due to integrated prophage elements and Salmonella genomic island 1 encoding antibiotic resistance genes. Thirteen isolates of Salmonella serovar Typhimurium DT104 with different pulsed-field gel electrophoresis (PFGE) profiles were analyzed by using multilocus sequence typing (MLST), plasmid profiling, hybridization to a pan-Salmonella DNA microarray, and prophage-based multiplex PCR. All the isolates belonged to a single MLST type, sequence type ST19. Microarray data demonstrated that the gene contents of the 13 DT104 isolates were remarkably conserved. The PFGE DNA fragment size differences in these isolates could be explained to a great extent by differences in the prophage and plasmid contents. Thus, here the nature of variation in different Salmonella serovar Typhimurium DT104 isolates is further defined at the gene and whole-genome levels, illustrating how this phage type evolves over time.     

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types

AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types. METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE). The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%). LPS profiling was able to discriminate between isolates of identical PFGE type. Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10. All 10 outbreaks were identical by PFGE analysis. CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles. The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS. SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations.     

Induction and resuscitation of viable but nonculturable Salmonella enterica serovar typhimurium DT104

Salmonella enterica serovar Typhimurium DT104 11601 was tested for its ability to maintain viability in minimal, chemically defined solutions. Periodic monitoring of growth and survival in microcosms of different ion concentrations, maintained at various temperatures, showed a gradual decline in culturable organisms ( approximately 235 days) at 5 degrees C. Organisms maintained at a higher temperature (21 degrees C) showed continuous, equivalent CFU per milliliter ( approximately 10(6)) up to 400 days after inoculation. Fluorescence microscopy with Baclight revealed that nonculturable cells were actually viable, while observations with scanning electron microscopy showed that the cells had retained their structural integrity. Temperature upshift (56 degrees C +/- 0.5, 15 s) of the nonculturable organisms (5 degrees C) in Trypticase soy broth followed by immediate inoculation onto Trypticase soy agar (TSA) gave evidence of resuscitation. Interestingly, S. enterica serovar Typhimurium DT104 from the microcosms at either 5 degrees C (1 to 200 days) or 21 degrees C (1 to 250 days) did not show enhanced growth after intermittent inoculation onto catalase-supplemented TSA. Furthermore, cells from 21 degrees C microcosms exposed to oxidative and osmotic stress showed greater resistance to stresses over increasing times of exposure than did recently grown cells. It is possible that the exceptional survivability and resilience of this particular strain may in part reflect the growing importance of this multidrug-resistant organism, in general, as a cause of intestinal disease in humans. The fact that S. enterica serovar Typhimurium DT104 11601 is capable of modifying its physiological characteristics, including entry into and recovery from the viable but nonculturable state, suggests the overall possibility that S. enterica serovar Typhimurium DT104 may be able to respond uniquely to various adverse environmental conditions.     

Genomic relationship of Salmonella enterica serovar typhimurium DT104 isolates from Korea and the United States

Salmonella enterica serovar Typhimurium DT104 (Salmonella Typhimurium DT104 or DT104) has been emerging as a common pathogen for human in Korea since 1997. In order to compare the genomic relationship and to search for the dominant strains in Korea, we conducted pulsed-field gel electrophoresis (PFGE) and IS200 fingerprinting of 25 epidemiological unrelated isolates from human and animals from Korea and cattle from America. Two Salmonella Typhimurium DT104 isolates from human in Korea and all 8 isolates from American cattle had indistinguishable patterns from the PFGE and IS200 fingerprinting but multidrug-resistant Salmonella Typhimurium isolates, including DT104, from Korean animals had diverse genetic patterns. The data suggest that a dominant DT104 strain might have circulated between Korean and American cattle and that it had a high level of clonality.     

Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64

The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.     

Characterization of grvA, an antivirulence gene on the gifsy-2 phage in Salmonella enterica serovar typhimurium

The lambdoid phage Gifsy-2 contributes significantly to Salmonella enterica serovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene, sodCI, and other unidentified virulence factors. We have characterized the gene grvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes, grvA null mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-type grvA in single copy complemented this phenotype. However, grvA(+) on a multicopy plasmid also conferred the antivirulence phenotype. Neither a grvA null mutation nor the grvA(+) plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by the grvA null mutation and the grvA(+) plasmid required wild-type sodCI but was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such as sodCI and antivirulence factors such as grvA.     

Analysis of integrons in human isolates of Salmonella enterica serovar typhimurium isolated in the Slovak Republic

About 110 sporadic, epidemiologically unrelated Salmonella enterica serovar typhimurium strains isolated in the Slovak Republic were analyzed for the presence of integrons. Of these 110 examined strains, 47 were of definitive phage type DT104 and 63 were strains of various phage type, RDNC and untypeable, designated here as non-DT104 strains. All isolates were also tested for antimicrobial resistance to 10 antibiotics as well as for the presence of virulence plasmid. Of 63 non-DT104 strains, 15 isolates were multiple-resistant, independently from phage type, other strains were resistant to one, two or three drugs. Resistance to ampicillin, streptomycin, tetracycline and sulfisoxazole was most frequently observed. Among the DT104 isolates up 65.9% exhibited characteristic pentaresistance--ACSSuT phenotype. The integron content was studied in PCR experiments using a 5'-CS/3'-CS primer pair. Fourteen non-DT104 strains, independently from phage type, were found to carry integrons with amplicons 650-1900 bp in size. Thirty-six DT104 strains contained integrons of 1000 and 1200 bp and 31 of they exhibited the ACSSuT phenotype. No integron was found in 10 DT104 strains, which included strains mostly resistant only to streptomycin, tetracycline and sulfisoxazole. The majority of non-DT104 strains did not possess any integrons. Our findings show the widespread existence of both resistant and multiple-resistant epidemiologically unrelated Salmonella typhimurium strains and suggest that integrons contribute to this antimicrobial resistance. The presence of 90-kb virulence plasmid in the 54 non-DT104 and in the all DT104 strains was found.     

No genetic barriers between Salmonella enterica serovar typhimurium and Escherichia coli in SOS-induced mismatch repair-deficient cells

Conjugational crosses trigger SOS induction in Escherichia coli F(-) cells mated with Salmonella enterica serovar Typhimurium Hfr donors. Using an epigenetic indicator of SOS induction, we showed that a strong SOS response occurring in a subpopulation of mated mismatch repair-deficient cells totally abolishes genetic barriers between these two genera.     

Proteomic analysis of Salmonella enterica serovar typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhimurium inside macrophages

To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.     

Resuscitation by ferrioxamine E of stressed Salmonella enterica serovar typhimurium from soil and water microcosms

Storage of Salmonella enterica serovar Typhimurium strains in soil and water microcosms resulted in loss of culturability on standard plating media. Prior incubation in buffered peptone water supplemented with ferrioxamine E markedly extended the time that bacteria were recoverable by plating, except in the case of mutants deficient in ferrioxamine E uptake.     

Molecular characterization of the multidrug-resistant phage types Salmonella enterica serovar Typhimurium DT104, DT20A and DT120 strains in the Slovakia

An increase in the number of multidrug-resistant Salmonella enterica serovar Typhimurium strains (definitive phage type DT20a and DT120) as well as the occurrence of DT104 strains during 2003-2005 in Slovakia was documented. Based on the results of the molecular analysis we suggest that multidrug-resistant DT20a and DT120 phage types are more closely related to multidrug-resistant phage type, and that the occurrence is probably due to changes in the phage susceptibility of DT104. Continued surveillance and molecular analysis should be maintained to follow the spread of these new multidrug-resistant DT104 variants in animals and humans.     

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.