Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,[5],12:i:- of Food Origin in China

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more comprehensive and representative, providing valuable information for epidemiological studies, risk management, and public health strategies.     

Biofilm Formation by Salmonella Enterica Serovar 1,4,[5],12:i:- Portuguese Isolates: A Phenotypic,Genotypic, and Socio-geographic Analysis

Biofilm-forming ability is well established as an important virulence factor. However, there are no studies available regarding biofilm formation of Salmonella Typhimurium 1,4,[5],12:i:-, the new pandemic serovar in Europe. To address this problem, biofilm expression by Salmonella 1,4,[5],12:i:- was evaluated using 133 isolates from clinical, environmental and animal origins, collected in Portugal from 2006 to 2011. Biofilm detection was performed by phenotypic and genotypic methods, such growth characterization in agar and broth medium, optical density determination by microtiter assays and direct observation by fluorescent in situ hybridization. Biofilm-related genes adrA, csgD and gcpA were detected by PCR. A socio-geographic characterization of strains as biofilm producers was also performed. Results showed that biofilm formation in monophasic Salmonella is widely distributed in Portuguese isolates and could be one of the reasons for its dissemination in this country. Biofilm expression varies between locations, showing that isolates from some regions like Lisboa or Ponta Delgada have an increased ability to persist in the environment due to an enhanced biofilm production. Biofilm formation also varies between risk groups, with a higher prevalence in isolates from salmonellosis infections in women. Therefore, the analysis of the socio-geographic distribution of biofilm-forming bacteria should be considered for the establishment of more adequate regulatory measures or therapeutics regimens, especially important due to the continuous increase of infections caused by antimicrobial resistant microorganisms.     

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.     

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°C resulted in more rapid loss of viability than incubation at 21°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 μm 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.     

Oxidoreductases that Act as Conditional Virulence Suppressors in Salmonella enterica Serovar Typhimurium

In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.     

Suppression of Conformation-Compromised Mutants of Salmonella enterica Serovar Typhimurium MelB

The crystal structure of the Na⁺-coupled melibiose permease of Salmonella enterica serovar Typhimurium (MelB_St) demonstrates that MelB is a member of the major facilitator superfamily of transporters. Arg residues at positions 295, 141, and 363 are involved in interdomain interactions at the cytoplasmic side by governing three clusters of electrostatic/polar interactions. Insertion of (one at a time) Glu, Leu, Gln, or Cys at positions R295, R141, and R363, or Lys at position R295, inhibits active transport of melibiose to a level of 2 to 20% of the value for wild-type (WT) MelB_St, with little effect on binding affinities for both sugar and Na⁺. Interestingly, a spontaneous suppressor, D35E (periplasmic end of helix I), was isolated from the R363Q MelB_St mutant. Introduction of the D35E mutation in each of the mutants at R295, R141 (except R141E), or R363 rescues melibiose transport to up to 91% of the WT value. Single-site mutations for the pair of D35 and R175 (periplasmic end of helix VI) were constructed by replacing Asp with Glu, Gln, or Cys and R175 with Gln, Asn, or Cys. All mutants with mutations at R175 are active, indicating that a positive charge at R175 is not necessary. Mutant D35E shows reduced transport; D35Q and D35C are nearly inactivated. Surprisingly, the D35Q mutation partially rescues both R141C and R295Q mutations. The data support the idea that Arg at position 295 and a positive charge at positions 141 and 363 are required for melibiose transport catalyzed by MelB_St, and their mutation inhibits conformational cycling, which is suppressed by a minor modification at the opposite side of the membrane.     

Characterization of an Acid-Inducible Sulfatase in Salmonella enterica Serovar Typhimurium

Sulfatases of enteric bacteria can provide access to heavily sulfated mucosal glycans. In this study, we show that aslA (STM0084) of Salmonella enterica serovar Typhimurium LT2 encodes a sulfatase that requires mildly acidic pH for its expression and activity. AslA is not regulated by sulfur compounds or tyramine but requires the EnvZ-OmpR and PhoPQ regulatory systems, which play an important role in pathogenesis.     

Epidemiology of a Salmonella enterica subsp. enterica Serovar Typhimurium Strain Associated with a Songbird Outbreak

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.     

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 10⁴ CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 10³ CFU/g.     

Effect of Environmental Temperatures on Proteome Composition of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium (STM) is a major cause of gastroenteritis and transmitted by consumption of contaminated food. STM is associated to food originating from animals (pork, chicken, eggs) or plants (vegetables, fruits, nuts, and herbs). Infection of warm-blooded mammalian hosts by STM and the underlying complex regulatory network of virulence gene expression depend on various environmental conditions encountered in hosts. However, less is known about the proteome and possible regulatory networks for gene expression of STM outside the preferred host. Nutritional limitations and changes in temperature are the most obvious stresses outside the native host. Thus, we analyzed the proteome profile of STM grown in rich medium (LB medium) or minimal medium (PCN medium) at temperatures ranging from 8 °C to 37 °C. LB medium mimics the nutritional rich environment inside the host, whereas minimal PCN medium represents nutritional limitations outside the host, found during growth of fresh produce (field conditions). Further, the range of temperatures analyzed reflects conditions within natural hosts (37 °C), room temperature (20 °C), during growth under agricultural conditions (16 °C and 12 °C), and during food storage (8 °C). Implications of altered nutrient availability and growth temperature on STM proteomes were analyzed by HPLC/MS-MS and label-free quantification. Our study provides first insights into the complex adaptation of STM to various environmental temperatures, which allows STM not only to infect mammalian hosts but also to enter new infection routes that have been poorly studied so far. With the present dataset, global virulence factors, their impact on infection routes, and potential anti-infective strategies can now be investigated in detail. Especially, we were able to demonstrate functional flagella at 12 °C growth temperature for STM with an altered motility behavior.     

Selection of Orphan Rhs Toxin Expression in Evolved Salmonella enterica Serovar Typhimurium

Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic “orphan” toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the “main” rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.     

Curcumin Increases the Pathogenicity of Salmonella enterica Serovar Typhimurium in Murine Model

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer''s patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin''s mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks.     

Receptor Diversity and Host Interaction of Bacteriophages Infecting Salmonella enterica Serovar Typhimurium

Background

Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated.

Methodology/Principal Findings

Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B₁₂ uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain.

Conclusions/Significance

In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.