RESUMO
In this study, the changes in the global proteome of Salmonella in response to desiccation and thermal treatment were investigated by using an iTRAQ multiplex technique. A Salmonella enterica serovar Typhimurium strain was dried, equilibrated at high (1.0) and low (0.11) water activity (aw), and thermally treated at 75°C. The proteomes were characterized after every treatment. The proteomes of the different treatments differed in the expression of 175 proteins. On the basis of their proteomic expression profiles, the samples were clustered into two major groups, namely, "dry" samples and "moist" samples. The groups had different levels of proteins involved in DNA synthesis and transcription and in metabolic reactions, indicating that cells under either of the aw conditions need to strictly control energy metabolism, the rate of replication, and protein synthesis. The proteins with higher expression levels in moist samples were flagellar proteins (FlgEFGH), membrane proteins, and export systems (SecF, SecD, the Bam complex), as well as stress response proteins, suggesting that rehydration can trigger stress responses in moist cells. Dry samples had higher levels of ribosomal proteins, indicating that ribosomal proteins might be important for additional regulation of the cellular response, even when the synthesis of proteins is slowed down. At both aws, no differences in protein expression were observed between the thermally treated samples and the nonheated cells. In conclusion, our study indicates that the preadaptation to a dry condition was linked to increased thermal tolerance, while reversion from a dry state to a moist state induced a significant change in protein expression, possibly linked to the observed loss of thermal tolerance.IMPORTANCESalmonella enterica is able to survive in dry environments for very long periods. While it is well known that the initial exposure to desiccation is fundamental to trigger thermal tolerance in this organism, the specific physiological and molecular processes involved in this cross-protection phenomenon have not been fully characterized. Several studies have focused on the low-aw transcriptome of this pathogen when inoculated in different food matrices or on abiotic surfaces, but proteomic analyses have not been reported in the literature. Our study investigated the changes in proteomic expression in Salmonella enterica serovar Typhimurium during desiccation, exposure to low aw, and thermal treatment. A better knowledge of the systems involved in the response to desiccation and thermal tolerance, as well as a better understanding of their interplay, is fundamental to identify the most effective combination of interventions to prevent Salmonella's contamination of foods.
Assuntos
Dessecação , Salmonella typhimurium/fisiologia , Termotolerância , Água/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , ProteômicaRESUMO
Here we investigated whether Salmonella enterica serovar Typhimurium ATCC 14028 was capable of internalizing in peanut seedpods and plants when exposed to inoculated soil and the edaphic factors that influenced uptake. Intact dry Virginia (DV) and fresh green Virginia (GV) seedpods were exposed to soil containing 6.5 Log (CFU/g) Salmonella under different soil moisture conditions. Internalization of S. Typhimurium into peanut plants germinated in inoculated soil was also examined with and without Bradyrhizobium (Arachis) sp.NC92. Salmonella counts recovered from GV seedpods were on average of 2.0 Log (CFU/pod) less than those recovered from DV seedpods. The internalization in DV pods was only observed at soil water content of 15% or greater in a loamy sand soil. S. Typhimurium was detected inside peanut plant tissues during most testing times. Cells were recovered from stem samples (3.5 Log CFU/g) at greater levels than it was observed for root (2.6 Log CFU/g) and leaf (1.7 Log CFU/g) samples. Overall, recovery of Salmonella from stem, root, and leaf samples were lower when B. NC92 was inoculated on seeds before sowing, but this trend was not significant. Our observations suggest possible routes of contamination of Salmonella into peanut products from soil.
Assuntos
Arachis/microbiologia , Salmonella typhimurium/fisiologia , Sementes/microbiologia , Arachis/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Sementes/crescimento & desenvolvimento , Microbiologia do SoloRESUMO
Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.
Assuntos
Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Lactuca/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Transcrição Gênica , Aderência Bacteriana , Biofilmes , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/metabolismo , Contaminação de Alimentos/análise , Perfilação da Expressão GênicaRESUMO
Escherichia coli O157 is a foodborne pathogen that can be transmitted by contaminated ground beef and is shed naturally in cattle feces. Recent reports indicated that feeding distillers' grains (DG) to cattle increased fecal shedding and prevalence of E. coli O157. In Minnesota, feeding DG with solubles (DGS) to livestock became widespread within the last 10 years, but there is no report about the prevalence of E. coli O157 in beef cattle in this state. This study was undertaken to survey the fecal prevalence of E. coli O157 in cattle fed diets containing DG and its association with environmental conditions and management practices. Fecal samples were collected from three feedlots during a 1-year period. All animals in those feedlots were fed different DGS levels. E. coli O157 presence was determined using a combination of enrichment, immunomagnetic separation, plating onto sorbitol MacConkey agar, and confirmation of isolates by immunoassay and multiplex virulence genes polymerase chain reaction analysis. Overall, E. coli O157 was confirmed in 9.7% of samples. Prevalence during summer was 30% and declined to less than 10% the rest of the year. In animals grouped by dietary DGS concentration, no significant difference in prevalence (12.0 and 5.5%) was detected between the low and the high average groups (less and more than 20%). Previous feeding of DGS before arriving to the feedlot also had no influence on fecal prevalence. The presence of several interacting variables, uncontrolled in a real-life feedlot environment, was the likely reason for our observation and suggested that at the levels studied, DGS had no effect on the STEC O157 prevalence in cattle populations.
Assuntos
Ração Animal , Criação de Animais Domésticos/métodos , Bovinos/microbiologia , Grão Comestível , Escherichia coli O157/crescimento & desenvolvimento , Resíduos , Bebidas Alcoólicas/economia , Ração Animal/efeitos adversos , Ração Animal/economia , Criação de Animais Domésticos/economia , Animais , Derrame de Bactérias , Biocombustíveis/economia , Destilação , Grão Comestível/efeitos adversos , Escherichia coli O157/classificação , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Etanol/metabolismo , Fezes/microbiologia , Feminino , Fermentação , Indústria de Processamento de Alimentos/economia , Masculino , Indústria de Embalagem de Carne/economia , Indústria de Embalagem de Carne/métodos , Minnesota , Tipagem Molecular , Estações do Ano , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Resíduos/efeitos adversos , Resíduos/economiaRESUMO
An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.
Assuntos
Escherichia coli K12/genética , Escherichia coli O157/genética , Lactuca/microbiologia , Folhas de Planta/microbiologia , Transcriptoma , Aderência Bacteriana , Escherichia coli K12/fisiologia , Escherichia coli O157/fisiologia , Análise em MicrossériesRESUMO
Several reports have indicated that the thermal tolerance of Salmonella at low-water activity increases significantly, but information on the impact of diverse food matrices is still scarce. The goal of this research was to determine the kinetic parameters (decimal reduction time, D; time required for the first decimal reduction, δ) of thermal resistance of Salmonella in a previously cooked low water activity food. Commercial toasted oats cereal (TOC) was used as the food model, with or without sucrose (25%) addition. TOC samples were inoculated with 108 CFU/mL of a single strain of one of three Salmonella serovars (Agona, Tennessee, Typhimurium). TOC samples were ground and equilibrated to aw values of 0.11, 0.33 and 0.53, respectively. Ground TOC was heated at temperatures between 65 °C and 105 °C and viable counts were determined over time (depending on the temperature for up to 6 h). Death kinetic parameters were determined using linear and Weibull regression models. More than 70% of Weibull's adjusted regression coefficients (Radj2) and only 38% of the linear model's Radj2 had values greater than 0.8. For all serovars, both D and δ values increased consistently at a 0.11 aw compared to 0.33 and 0.53. At 0.33 aw, the δ values for Typhimurium, Tennessee and Agona were 0.55, 1.01 and 2.87, respectively, at 85 °C, but these values increased to 65, 105 and 64 min, respectively, at 0.11 aw. At 100 °C, δ values were 0.9, 5.5 and 2.3 min, respectively, at 0.11 aw. The addition of sucrose resulted in a consistent reduction of eight out of nine δ values determined at 0.11 aw at 85, 95 and 100 °C, but this trend was not consistent at 0.33 and 0.53 aw. The Z values (increase of temperature required to decrease δ-value one log) were determined with modified δ values for a fixed ß (a fitting parameter that describes the shape of the curve), and ranged between 8.9 °C and 13.4 °C; they were not influenced by aw, strain or sugar content. These findings indicated that in TOC, high thermal tolerance was consistent among serovars and thermal tolerance was inversely dependent on aw.
RESUMO
Enterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, virulence factors and MGEs were profiled in a set of 1273 E. faecium genomes from two disparate geographic locations (in the UK and Canada) from a range of agricultural, clinical and associated habitats. Neither lineages of E. faecium, type A and B, nor MGEs are constrained by geographic proximity, but our results show evidence of a strong association of many profiled genes and MGEs with habitat. Many features were associated with a group of clinical and municipal wastewater genomes that are likely forming a new human-associated ecotype within type A. The evolutionary dynamics of E. faecium make it a highly versatile emerging pathogen, and its ability to acquire, transmit and lose features presents a high risk for the emergence of new pathogenic variants and novel resistance combinations. This study provides a workflow for MGE-centric surveillance of AMR in Enterococcus that can be adapted to other pathogens.
Assuntos
Anti-Infecciosos , Enterococcus faecium , Saúde Única , Enterococcus faecium/genética , Humanos , Fatores de Virulência/genética , Águas ResiduáriasRESUMO
Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Salmonella typhimurium/fisiologia , Transativadores/biossíntese , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/genética , Imunoprecipitação da Cromatina , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/biossíntese , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Genes Bacterianos , Ilhas Genômicas , Camundongos , Camundongos Endogâmicos C3H , Óperon , Ligação Proteica , Proteínas Repressoras/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Análise de Sobrevida , Fatores de Transcrição/biossíntese , VirulênciaRESUMO
BACKGROUND: The Ferric uptake regulator (Fur) is a transcriptional regulator that controls iron homeostasis in bacteria. Although the regulatory role of Fur in Escherichia coli is well characterized, most of the studies were conducted under routine culture conditions, i.e., in ambient oxygen concentration. To reveal potentially novel aspects of the Fur regulon in Salmonella enterica serovar Typhimurium under oxygen conditions similar to that encountered in the host, we compared the transcriptional profiles of the virulent wild-type strain (ATCC 14028s) and its isogenic Δfur strain under anaerobic conditions. RESULTS: Microarray analysis of anaerobically grown Δfur S. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, sodB, occurred via small RNAs (i.e., the ryhB homologs, rfrA and rfrB) with the aid of the RNA chaperone Hfq. The transcription of sodA was increased in Δfur; however, the enzyme was inactive due to the incorporation of iron instead of manganese in SodA. Additionally, in Δfur, the expression of the gene coding for the ferritin-like protein (ftnB) was down-regulated, while the transcription of the gene coding for the nitric oxide (NO·) detoxifying flavohemoglobin (hmpA) was up-regulated. The promoters of ftnB and hmpA do not contain recognized Fur binding motifs, which indicated their probable indirect regulation by Fur. However, Fur activation of ftnB was independent of Fnr. In addition, the expression of the gene coding for the histone-like protein, H-NS (hns) was increased in Δfur. This may explain the observed down-regulation of the tdc operon, responsible for the anaerobic degradation of threonine, and ftnB in Δfur. CONCLUSIONS: This study determined that Fur is a positive factor in ftnB regulation, while serving to repress the expression of hmpA. Furthermore, Fur is required for the proper expression and activation of the antioxidant enzymes, FeSOD and MnSOD. Finally, this work identified twenty-six new targets of Fur regulation, and demonstrates that H-NS repressed genes are down-regulated in Δfur.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxigênio/metabolismo , Regulon , Proteínas Repressoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Ferro/metabolismo , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In Escherichia coli, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in E. coli is well characterized; however, little is known about its role in anaerobically grown S. Typhimurium. RESULTS: We compared the transcriptional profiles of the virulent wild-type (WT) strain (ATCC 14028s) and its isogenic arcA mutant grown under anaerobic conditions. We found that ArcA directly or indirectly regulates 392 genes (8.5% of the genome); of these, 138 genes are poorly characterized. Regulation by ArcA in S. Typhimurium is similar, but distinct from that in E. coli. Thus, genes/operons involved in core metabolic pathways (e.g., succinyl-CoA, fatty acid degradation, cytochrome oxidase complexes, flagellar biosynthesis, motility, and chemotaxis) were regulated similarly in the two organisms. However, genes/operons present in both organisms, but regulated differently by ArcA in S. Typhimurium included those coding for ethanolamine utilization, lactate transport and metabolism, and succinate dehydrogenases. Salmonella-specific genes/operons regulated by ArcA included those required for propanediol utilization, flagellar genes (mcpAC, cheV), Gifsy-1 prophage genes, and three SPI-3 genes (mgtBC, slsA, STM3784). In agreement with our microarray data, the arcA mutant was non-motile, lacked flagella, and was as virulent in mice as the WT. Additionally, we identified a set of 120 genes whose regulation was shared with the anaerobic redox regulator, Fnr. CONCLUSION(S): We have identified the ArcA regulon in anaerobically grown S. Typhimurium. Our results demonstrated that in S. Typhimurium, ArcA serves as a transcriptional regulator coordinating cellular metabolism, flagella biosynthesis, and motility. Furthermore, ArcA and Fnr share in the regulation of 120 S. Typhimurium genes.
Assuntos
Regulação Bacteriana da Expressão Gênica , Regulon , Proteínas Repressoras/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/genética , Anaerobiose , Animais , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Proteínas Repressoras/genética , Salmonella typhimurium/metabolismoRESUMO
MALDI-TOF is a chemistry analytical tool that has recently been deployed in the identification of microorganisms isolated from nosocomial environments. Its use in diagnostics has been extremely advantageous in terms of cost effectiveness, sample preparation easiness, turn-around time and result analysis accessibility. In the dairy industry, where mastitis causes great financial losses, a rapid diagnostic method such as MALDI-TOF could assist in the control and prevention program of mastitis, in addition to the sanitation and safety level of the dairy farms and processing facility. However, the diagnostic strengths and limitations of this test method require further understanding. In the present study, we prospectively compared MALDI-TOF MS to conventional 16S rDNA sequencing method for the identification of pathogens recovered from milk associated with clinical and subclinical bovine mastitis cases. Initially, 810 bacterial isolates were collected from raw milk samples over a period of three months. However, only the isolates (481) having both 16S rDNA sequencing and MALDI-TOF identification were included in the final phase of the study. Among the 481 milk isolates, a total of 26 genera (12 g-postive and 14 g-negative), including 71 different species, were taxonomically charecterized by 16S rDNA at the species level. Comparatively, MALDI-TOF identified 17 genera (9 g-positive and 8 g-negative) and 33 differernt species. Overall, 445 (93%) were putatively identified to the genus level by MALDI-TOF MS and 355 (74%) were identified to the species level, but no reliable identification was obtained for 16 (3.3%), and 20 (4.2%) discordant results were identified. Future studies may help to overcome the limitations of the MALDI database and additional sample preparation steps might help to reduce the number of discordances in identification. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional identification methods for common mastitis pathogens, routinely isolated from raw milk. Thus it's adoption will strengthen the capacity, quality, and possibly the scope of diagnostic services to support the dairy industry.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas , Mastite Bovina , Leite/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Bovinos , Feminino , Mastite Bovina/diagnóstico , Mastite Bovina/microbiologia , RNA Ribossômico 16S/isolamento & purificaçãoRESUMO
A nettle (Urtica dioica) extract shows in vitro inhibition of several key inflammatory events that cause the symptoms of seasonal allergies. These include the antagonist and negative agonist activity against the Histamine-1 (H(1)) receptor and the inhibition of mast cell tryptase preventing degranulation and release of a host of pro-inflammatory mediators that cause the symptoms of hay fevers. The nettle extract also inhibits prostaglandin formation through inhibition of Cyclooxygenase-1 (COX-1), Cyclooxygenase-2 (COX-2), and Hematopoietic Prostaglandin D(2) synthase (HPGDS), central enzymes in pro-inflammatory pathways. The IC(50) value for histamine receptor antagonist activity was 251 (+/-13) microg mL(-1) and for the histamine receptor negative agonist activity was 193 (+/-71) microg mL(-1). The IC(50) values for inhibition of mast cell tryptase was 172 (+/-28) microg mL(-1), for COX-1 was 160 (+/-47) microg mL(-1), for COX-2 was 275 (+/-9) microg mL(-1), and for HPGDS was 295 (+/-51) microg mL(-1). Through the use of DART TOF-MS, which yields exact masses and relative abundances of compounds present in complex mixtures, bioactives have been identified in nettle that contribute to the inhibition of pro-inflammatory pathways related to allergic rhinitis. These results provide for the first time, a mechanistic understanding of the role of nettle extracts in reducing allergic and other inflammatory responses in vitro.
Assuntos
Antagonistas dos Receptores Histamínicos/farmacologia , Mediadores da Inflamação/farmacologia , Extratos Vegetais/farmacologia , Rinite Alérgica Perene/tratamento farmacológico , Urtica dioica/química , Linhagem Celular , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Espectrometria de Massas , Triptases/antagonistas & inibidoresRESUMO
Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella's cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella's survival during desiccation.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Fatores de Virulência/genética , Adaptação Fisiológica/genética , Proteínas de Bactérias/metabolismo , Dessecação , Deleção de Genes , Perfilação da Expressão Gênica , Microscopia Eletrônica de Varredura , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Estresse Fisiológico/genética , Transcriptoma , Fatores de Virulência/deficiênciaRESUMO
Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate.
RESUMO
Leafy green vegetables have been identified as a source of foodborne illnesses worldwide over the past decade. Human enteric pathogens, such as Escherichia coli O157:H7 and Salmonella, have been implicated in numerous food poisoning outbreaks associated with the consumption of fresh produce. An understanding of the mechanisms responsible for the establishment of pathogenic bacteria in or on vegetable plants is critical for understanding and ameliorating this problem as well as ensuring the safety of our food supply. While previous studies have described the growth and survival of enteric pathogens in the environment and also the risk factors associated with the contamination of vegetables, the molecular events involved in the colonization of fresh produce by enteric pathogens are just beginning to be elucidated. This review summarizes recent findings on the interactions of several bacterial pathogens with leafy green vegetables. Changes in gene expression linked to the bacterial attachment and colonization of plant structures are discussed in light of their relevance to plant-microbe interactions. We propose a mechanism for the establishment and association of enteric pathogens with plants and discuss potential strategies to address the problem of foodborne illness linked to the consumption of leafy green vegetables.
Assuntos
Enterobacteriaceae/fisiologia , Contaminação de Alimentos , Inocuidade dos Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Verduras/microbiologia , Animais , Enterobacteriaceae/crescimento & desenvolvimento , Escherichia coli O157/fisiologia , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Insetos Vetores/microbiologia , Folhas de Planta/microbiologia , Sementes/microbiologia , Microbiologia do SoloRESUMO
Lettuce is the fresh leafy vegetable most frequently involved in foodborne disease outbreaks. Human bacterial pathogens may be experimentally internalized into lettuce plants, but the occurrence of natural microflora inside lettuce leaves has not been elucidated. To characterize the endophytic microorganism residing in commercial lettuce leaves, two separate studies were conducted. First, a total of 30 and 25 heads of romaine and red leaf lettuce, respectively, served as the source of individual leaves which were surface sterilized, stomached, enriched in BHI broth for 24h and plated onto BHI agar for non-selective isolation of internalized microorganism. In a separate survey, 80 heads of each of the two types of lettuce were similarly processed, except that GN broth and MacConkey agar (MCA) were used for isolation of Gram negative bacteria. Thirty-eight out of 100 leaves were positive for internalized microorganisms, and Bacillus, Pseudomonas and Pantoea were the genera most frequently found in both types of lettuce. Members of the genus Erwinia were isolated from romaine lettuce only. In the second study, 21 and 60% of romaine and red leaf lettuce heads, respectively, had internalized bacteria capable of growing on MCA. Among the Gram negative strains, Pseudomonas and Pantoea genera were most frequently isolated. Enterobacter isolates were obtained from three red leaf samples. In summary, spore-forming bacteria and traditional epiphytic bacterial genera were frequently detected in surface-sterilized commercial lettuce leaves. Despite the common occurrence of internalized bacteria, only Enterobacter was related to Escherichia coli O157:H7 and Salmonella.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Microbiologia de Alimentos , Lactuca/microbiologia , Bacillus/isolamento & purificação , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Enterobacter/isolamento & purificação , Escherichia coli O157/isolamento & purificação , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Pantoea/isolamento & purificação , Folhas de Planta/microbiologia , Pseudomonas/isolamento & purificação , Salmonella/isolamento & purificaçãoRESUMO
In a previous in vitro study, the standardized turmeric extract, HSS-888, showed strong inhibition of Aß aggregation and secretion in vitro, indicating that HSS-888 might be therapeutically important. Therefore, in the present study, HSS-888 was evaluated in vivo using transgenic 'Alzheimer' mice (Tg2576) over-expressing Aß protein. Following a six-month prevention period where mice received extract HSS-888 (5mg/mouse/day), tetrahydrocurcumin (THC) or a control through ingestion of customized animal feed pellets (0.1% w/w treatment), HSS-888 significantly reduced brain levels of soluble (â¼40%) and insoluble (â¼20%) Aß as well as phosphorylated Tau protein (â¼80%). In addition, primary cultures of microglia from these mice showed increased expression of the cytokines IL-4 and IL-2. In contrast, THC treatment only weakly reduced phosphorylated Tau protein and failed to significantly alter plaque burden and cytokine expression. The findings reveal that the optimized turmeric extract HSS-888 represents an important step in botanical based therapies for Alzheimer's disease by inhibiting or improving plaque burden, Tau phosphorylation, and microglial inflammation leading to neuronal toxicity.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/uso terapêutico , Extratos Vegetais/uso terapêutico , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Amiloidose/tratamento farmacológico , Análise de Variância , Animais , Antioxidantes/farmacologia , Curcuma , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: The development of antiviral drugs has provided crucial new means to mitigate or relieve the debilitating effects of many viral pathogens. Regular use of these drugs has led to generation of resistant strains, making the control of many viral infections very difficult, particularly in HIV-seropositive and AIDS patients. A rich source for the discovery of new HIV infection inhibitors has been, and continues to be, the 'mining' of the large diversity of compounds already available in nature, and specifically those from botanical extracts. METHODS: Using a newly developed direct binding assay with mass spectrometry technology (direct analysis in real-time time-of-flight mass spectrometry), we were able to show that compounds present in extracts of elderberry, cinnamon and green tea bind to and block HIV type-1 (HIV-1) infection in target cells. RESULTS: The compounds that blocked HIV-1 infection were flavonoids and A-type proanthocyanidins. The 50% inhibitory concentration values of these extracts ranged from 0.5 to 201 microg/ml for four different HIV-1 serotypes. Interaction matrices with the elderberry extract and enfuvirtide, a peptide HIV-1 fusion inhibitor, revealed significant super additive effects. This indicates that the compounds in elderberry that prevent HIV-1 infection are likely to bind to viral glycoproteins other than gp41 (the binding site for enfuvirtide). CONCLUSIONS: Optimized elderberry, green tea and cinnamon extracts rich in certain flavonoid compounds were shown to block HIV-1 entry and infection in GHOST cells. As such, these types of botanical extracts could provide a starting point for the development of possible safe and reliable cotherapies for HIV-1-positive individuals, as well as for the identification of new small molecules as leading drug candidates for HIV-1 therapeutics and microbicides.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Extratos Vegetais/farmacologia , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/toxicidade , Sítios de Ligação , Camellia sinensis/química , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Cinnamomum zeylanicum/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Sinergismo Farmacológico , Enfuvirtida , Proteína gp41 do Envelope de HIV/farmacologia , Infecções por HIV/prevenção & controle , HIV-1/metabolismo , Humanos , Espectrometria de Massas , Fragmentos de Peptídeos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Extratos Vegetais/toxicidade , Reprodutibilidade dos Testes , Sambucus/química , Fatores de Tempo , Vírion/efeitos dos fármacos , Vírion/metabolismo , Vírion/fisiologiaRESUMO
Rice bran, the outer bran and germ of the kernel and a by-product of rice milling, is rich in phytonutrients but has been underutilized because of lipid content instability. New methods for the processing of rice bran have yielded a stabilized form that is increasingly used in foods and dietary supplements. Recent studies have documented a role for stabilized rice bran (SRB) in treating diabetes and arthritis, although little is known of the bioactive compounds that impart these health benefits. Here we characterize the chemical composition of three extracts of SRB and identify the functional bioactives contributing to the inhibitory properties against three key pro-inflammatory enzymes (cyclooxygenase [COX] 1, COX2, and 5-lipoxygenase [5-LOX]) that control the inflammatory cascade involved in impaired joint health, pain, and arthritis. One extract (SRB-AI) demonstrated significant COX1 and COX2 inhibitory activities with 50% inhibitory concentration (IC(50)) values for COX1 and COX2 of 305 and 29 microg/mL, respectively, but no 5-LOX inhibition. The second extract (SRB-AII) inhibited COX1, COX2, and 5-LOX with IC(50) values of 310, 19, and 396 microg/mL, respectively. The third extract (SRB-AIII), a blend of SRB-AI and SRB-AIII, inhibited COX1, COX2, and 5-LOX with respective IC(50) values of 48, 11, and 197 microg/mL. Analysis of the extracts by direct analysis in real time time of flight-mass spectrometry revealed that SRB-AI, SRB-AII, and SRB-AIII contain over 620, 770, and 810 compounds, respectively. Of these, 17 were identified as key bioactives for COX and/or LOX inhibition. These SRB extracts have applications for functional foods and dietary supplements for control of inflammation and joint health.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Lipoxigenase/farmacologia , Oryza/química , Extratos Vegetais/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/isolamento & purificação , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/isolamento & purificação , Relação Dose-Resposta a Droga , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , SementesRESUMO
A ionization technique in mass spectrometry called Direct Analysis in Real Time Mass Spectrometry (DART TOF-MS) coupled with a Direct Binding Assay was used to identify and characterize anti-viral components of an elderberry fruit (Sambucus nigra L.) extract without either derivatization or separation by standard chromatographic techniques. The elderberry extract inhibited Human Influenza A (H1N1) infection in vitro with an IC(50) value of 252+/-34 microg/mL. The Direct Binding Assay established that flavonoids from the elderberry extract bind to H1N1 virions and, when bound, block the ability of the viruses to infect host cells. Two compounds were identified, 5,7,3',4'-tetra-O-methylquercetin (1) and 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexanecarboxylate (2), as H1N1-bound chemical species. Compound 1 and dihydromyricetin (3), the corresponding 3-hydroxyflavonone of 2, were synthesized and shown to inhibit H1N1 infection in vitro by binding to H1N1 virions, blocking host cell entry and/or recognition. Compound 1 gave an IC(50) of 0.13 microg/mL (0.36 microM) for H1N1 infection inhibition, while dihydromyricetin (3) achieved an IC(50) of 2.8 microg/mL (8.7 microM). The H1N1 inhibition activities of the elderberry flavonoids compare favorably to the known anti-influenza activities of Oseltamivir (Tamiflu; 0.32 microM) and Amantadine (27 microM).