E. coli K-12 and EHEC Genes Regulated by SdiA

Background: Escherichia and Salmonella encode SdiA, a transcription factor of the LuxR family that regulates genes in response to N-acyl homoserine lactones (AHLs) produced by other species of bacteria. E. coli genes that change expression in the presence of plasmid-encoded sdiA have been identified by several labs. However, many of these genes were identified by overexpressing sdiA on a plasmid and have not been tested for a response to sdiA produced from its natural position in the chromosome or for a response to AHL. Methodology/Principal Findings: We determined that two important loci reported to respond to plasmid-based sdiA, ftsQAZ and acrAB, do not respond to sdiA expressed from its natural position in the chromosome or to AHLs. To identify genes that are regulated by chromosomal sdiA and/or AHLs, we screened 10,000 random transposon-based luciferase fusions in E. coli K-12 and a further 10,000 in E. coli O157:H7 for a response to AHL and then tested these genes for sdiAdependence. We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA. Gene regulation by sdiA of E. coli is only partially dependent upon AHL. Conclusions/Significance: The genes of E. coli that respond to plasmid-based expression of sdiA are largely different than those that respond to chromosomal sdiA and/or AHL. This has significant implications for determining the true function o

Virulence of 32 Salmonella Strains in Mice

Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies

16S rRNA analysis provides evidence of biofilms on all components of three infected periprosthetic knees including permanent braided suture

Bacterial biofilms are a main etiological agent of periprosthetic joint infections (PJI), however it is unclear if biofilms colonize one or multiple components. Because biofilms can colonize a variety of surfaces, we hypothesized that biofilms would be present on all components. 16S rRNA gene sequencing analysis was used to identify bacteria recovered from individual components and non-absorbable suture material recovered from three PJI total knee revision cases. Bray-Curtis non-metric multidimensional scaling analysis revealed no significant differences in similarity when factoring component, material type, or suture vs. non-suture material, but did reveal significant differences in organism profile between patients (p &lt; 0.001) and between patients and negative controls (p &lt; 0.001). Confocal microscopy and a novel agar encasement culturing method also confirmed biofilm growth on a subset of components. While16S sequencing suggested that the microbiology was more complex than revealed by culture contaminating bacterial DNA generates a risk of false positives. This report highlights that biofilm bacteria may colonize all infected prosthetic components including braided suture material and provides further evidence that clinical culture can fail to sufficiently identify the full pathogen profile in PJI cases.<br/

Elution of antibiotics from poly(methyl methacrylate) bone cement after extended implantation does not necessarily clear the infection despite susceptibility of the clinical isolates

Chronic orthopedic infections are commonly caused by bacterial biofilms, which are recalcitrant to antibiotic treatment. In many cases, the revision procedure for periprosthetic joint infection or trauma cases includes the implantation of antibiotic-loaded bone cement to kill infecting bacteria via the elution of a strong local dose of antibiotic(s) at the site. While many studies have addressed the elution kinetics of both non-absorbable and absorbable bone cements both in vitro and in vivo, the potency of ALBC against pathogenic bacteria after extended implantation time is not clear. In this communication, we use two case studies, a Viridans streptococci infected total knee arthroplasty (TKA) and a MRSA-polymicrobial osteomyelitis of a distal tibial traumatic amputation (TA) to demonstrate that an antibiotic-loaded poly(methyl methacrylate) (ALPMMA) coated intermedullary rod implanted for 117 days (TKA) and three ALPMMA suture-strung beads implanted for 210 days (TA) retained killing ability against Pseudomonas aeruginosa and Staphylococcus aureus in vitro, despite different clinical efficacies. The TKA infection resolved and the patient progressed to an uneventful second stage. However, the TA infection only resolved after multiple rounds of debridement, IV vancomycin and removal of the PMMA beads and placement of vancomycin and tobramycin loaded calcium sulfate beads.<br/

Virulence of 32 Salmonella Strains in Mice

Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies

Yersinia enterocolitica Inhibits Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Cellular Uptake

Yersinia enterocolitica biovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely, Salmonella enterica serovar Typhimurium uses a T3SS encoded by Salmonella pathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake of S. Typhimurium and Y. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other. Y. enterocolitica reduces S. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using an S. Typhimurium SPI1 mutant alone. However, Y. enterocolitica had no effect on S. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells. Y. enterocolitica was also able to inhibit the invasion of epithelial and macrophage-like cells by Listeria monocytogenes. Y. enterocolitica mutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blocking S. Typhimurium uptake by epithelial cells. S. Typhimurium encodes a LuxR homolog, SdiA, which detects N-acylhomoserine lactones (AHLs) produced by Y. enterocolitica and upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating the S. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed by Y. enterocolitica

The Gold Star Journal, 2011 Edition

The Gold Star Journal showcases the excellence of every academic department at The Citadel through the publication of cross-disciplinary, nonfiction papers and creativity and ingenuity through the publication of photographs and photo stories. This edition's featured papers are: The Method Behind His Madness ; Race and Policing: An Assessment of Cultural Problems ; Racing Towards a Brave New World ; Wilfred Owen’s Brutal Honesty on the Wartime Experience ; The Evolution of Christianity in the American Society ; The Building of Braddock’s Road

<i>Salmonella</i> recovery and persistence in the feces.

<p>The left panel shows a cladogram that was inferred from core-genome SNPS using Maximum Parsimony. All positions containing gaps and missing data were eliminated. There were a total of 344642 positions of which 279065 were parsimony informative. The parsimony analysis was conducted using Mega5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036043#pone.0036043-Tamura1" target="_blank">[45]</a>. The middle panel shows <i>Salmonella</i> recovery from feces of surviving mice at 17 and 19 days post-infection. Dots represent individual measurements and the vertical line represents the median. The dotted line represents the 100 CFU/gm detection limit. The right panel represents the proportion of mice that were persistently infected with <i>Salmonella</i>. Black bars indicate that all mice met ERC. *One mouse met the ERC and was euthanized before the experiment was completed.</p

A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non eDNA networks in Pseudomonas aeruginosabiofilms

Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast enhancing stain using confocal laser scanning microscopy (CLSM) in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus, and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in P. aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but not did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool, and for diagnostic potential for biofilms in clinical samples.<br/

Salmonella enterica Serovar Typhimurium Can Detect Acyl Homoserine Lactone Production by Yersinia enterocolitica in Mice▿

LuxR-type transcription factors detect acyl homoserine lactones (AHLs) and are typically used by bacteria to determine the population density of their own species. Escherichia coli and Salmonella enterica serovar Typhimurium cannot synthesize AHLs but can detect the AHLs produced by other bacterial species using the LuxR homolog, SdiA. Previously we determined that S. Typhimurium did not detect AHLs during transit through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during transit through turtles colonized by Aeromonas hydrophila, leading to the hypothesis that SdiA is used for detecting the AHL production of other pathogens. In this report, we determined that SdiA is activated during the transit of S. Typhimurium through mice infected with the AHL-producing pathogen Yersinia enterocolitica. SdiA is not activated during transit through mice infected with a yenI mutant of Y. enterocolitica that cannot synthesize AHLs. However, activation of SdiA did not confer a fitness advantage in Yersinia-infected mice. We hypothesized that this is due to infrequent or short interactions between S. Typhimurium and Y. enterocolitica or that the SdiA regulon members do not function in mice. To test these hypotheses, we constructed an S. Typhimurium strain that synthesizes AHLs to mimic a constant interaction with Y. enterocolitica. In this background, sdiA+ S. Typhimurium rapidly outcompetes the sdiA mutant in mice. All known members of the sdiA regulon are required for this phenotype. Thus, all members of the sdiA regulon are functional in mice