Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104

Epidemic strain Salmonella typhimurium DT104 is characterized by various multiresistance patterns. At least some of the resistance genes are organized as integrons. Resistance genes of DT104 isolates can be efficiently transduced by P22-like phage ES18 and by phage PDT17 which is released by all DT104 isolates so far analyzed. Cotransduction tests demonstrate that the resistance genes, although not organized in a unique integron, are tightly clustered on the Salmonella chromosome. The spread of resistance genes in this strain by generalized transduction is discussed.     

Multiple Antibiotic Resistance (mar) Locus in Salmonella enterica Serovar Typhimurium DT104

In order to understand the role of the mar locus in Salmonella with regard to multiple antibiotic resistance, cyclohexane resistance, and outer membrane protein F (OmpF) regulation, a marA::gfp reporter mutant was constructed in an antibiotic-sensitive Salmonella enterica serovar Typhimurium DT104 background. Salicylate induced marA, whereas a number of antibiotics, disinfectants, and various growth conditions did not. Increased antibiotic resistance was observed upon salicylate induction, although this was shown to be by both mar-dependent and mar-independent pathways. Cyclohexane resistance, however, was induced by salicylate by a mar-dependent pathway. Complementation studies with a plasmid that constitutively expressed marA confirmed the involvement of mar in Salmonella with low-level antibiotic resistance and cyclohexane resistance, although the involvement of mar in down regulation of OmpF was unclear. However, marA overexpression did increase the expression of a ca. 50-kDa protein, but its identity remains to be elucidated. Passage of the marA::gfp reporter mutant with increasing levels of tetracycline, a method reported to select for mar mutants in Escherichia coli, led to both multiple-antibiotic and cyclohexane resistance. Collectively, these data indicate that low-level antibiotic resistance, cyclohexane resistance, and modulation of OMPs in Salmonella, as in E. coli, can occur in both a mar-dependent and mar-independent manner.     

The spread and antibiotic resistance of Salmonella enterica serotype typhimurium DT104 in Hungary

Comparison of phage types (PTs) determined by Felix and Callow's and Anderson's methods was performed testing 99 human strains of S. enterica serotype Typhimurium (S. typhimurium) isolated in Hungary. PT2 and PT2c--according to Felix-Callow--corresponded with Anderson's DT104 in case of 39 strains out of 40. Among 59 isolates belonging to other Felix-Callow's PTs only one strain was found which was DT 104. Similar unambiguous equalities could not be established between any other PTs comparing the two methods. The PTs of 17,877 human strains isolated between 1988 and 1999 were determined using Felix-Callow's method. On the basis of the above equality the emergence of DT104 could be followed retrospectively by means of the rate of PT2 and PT2c. The increase of DT104 began already in 1989, emerging first PT2c then PT2. It predominated since 1991 and it reached its maximum (78.3%) in 1999. The incidence of multiresistance among one of the groups of DT104 strains (Felix-Callow's PT2) was significantly higher in 1998 than the average of non-DT104 strains. The predominant R-type was ACST.     

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.     

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.     

Acid resistance variability among isolates of Salmonella enterica serovar Typhimurium DT104

AIMS: Acid resistance could be an indicator of virulence since acid resistant strains are able to better survive the human stomach passage and in macrophages. We studied the acid resistance of several Salmonella Typhimurium DT104 strains isolated from food and humans and identified cellular parameters contributing to the enhanced acid resistance of these isolates. METHODS AND RESULTS: Acid resistance was tested in 37 Salmonella enterica Typhimurium serovar DT104 (S. Typhimurium DT104) strains. Acid adaptation at pH 5 followed by exposure for 2 h at pH 2.5 in the 27 human, nine nonhuman, and in two reference strains, revealed strong variation of acid survival. After 2 h at pH 2.5 six strains of S. Typhimurium DT104 were considered high acid resistant as they displayed a level of survival >10%, 14 strains were considered intermediate acid resistant (level of survival was <10% and >0.01%) and 19 strains were considered low acid resistant (level of survival <0.01%). Six strains were selected for further studies and proteomics revealed a relatively high amount of phase 2 flagellin in an acid-sensitive strain and a relatively high amount of the beta component of the H(+)/ATPase in an acid-resistant strain. Two strains were slightly more heat resistant possibly as the result of increased levels of DnaK or GroEL. CONCLUSIONS: A significant difference could be detected between human and food isolates regarding their acid resistance; all high acid-resistant strains were human isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: S. Typhimurium DT104 is known for two decades and has a great impact on human health causing serious food-borne diseases. Our results suggest the existence of a positive correlation between acid resistance and pathogenicity in S. Typhimurium DT104 as all high acid-resistant strains were isolated from humans.     

Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25 degrees C after 3 days of incubation. Incubation at 37 degrees C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25 degrees C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.     

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.     

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enteritica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.     

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.     

Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar Typhimurium DT104

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37 degrees C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37 degrees C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25 degrees C than at 37 degrees C. Since the CP was shown to be produced at both 37 degrees C and 25 degrees C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.     

Capsular Polysaccharide Surrounds Smooth and Rugose Types of Salmonella enterica serovar Typhimurium DT104

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37°C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37°C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25°C than at 37°C. Since the CP was shown to be produced at both 37°C and 25°C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.     

Cloth-based hybridization array system for the detection of multiple antibiotic resistance genes in Salmonella enterica subsp. enterica serotype Typhimurium DT104

AIMS: A simple DNA macroarray system was developed for detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. METHODS AND RESULTS: A multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used to simultaneously amplify seven marker sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. This system provided sensitive detection of the different genetic markers in the S. Typhimurium DT104 genome, giving positive reactions with as few as 10 CFU, and the hybridizations were highly specific, with no reactions of amplicons with heterologous probes on the array. CONCLUSIONS: This cloth-based hybridization array system (CHAS) provides a simple, cost-effective tool for monitoring S. Typhimurium DT104 in foods and their production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The CHAS is a simple and cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the detection and characterization of food pathogens.     

Aspartic peptide hydrolases in Salmonella enterica serovar typhimurium

Two well-characterized enzymes in Salmonella enterica serovar Typhimurium and Escherichia coli are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of pepB pepE mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from E. coli, the product of the iadA gene. The other is the product of the serovar Typhimurium homolog of E. coli ybiK, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated iaaA (isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.     

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.     

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.     

Salmonella enterica Serovar Typhimurium DT104 Displays a Rugose Phenotype

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25°C after 3 days of incubation. Incubation at 37°C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25°C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.