A network of genes and at least two peptide signaling molecules tightly control when becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when is presented with competence signal peptides , including increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the genome with a screen to identify mutants that aberrantly expressed , coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g. ,, ,, ,, ,, but disclosed an additional 20 genes that were not previously competence-associated. The competence phenotypes of mutants were characterized, including using fluorescence microscopy to determine at which stage the mutants were impaired for activation. Among the novel genes studied were those implicated in cell division, sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in, while highlighting the effectiveness of using Tn-seq in to discover and study novel biological processes. acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of , while validating technologies that can rapidly advance our understanding of the physiology, biology and genetics of and related pathogens.
Rationale: Infectious disease remains the main cause of morbidity and mortality throughout the world.Of growing concern is the rising incidence of multidrug-resistant bacteria, derived from various selection pressures. Many of these bacterial infections are hospital-acquired and have prompted the Center for Disease Control and Prevention in 2019 to reclassify several pathogens as urgent threats, its most perilous assignment. Consequently, there is an urgent need to improve the clinical management of bacterial infection, via new methods to specifically identify bacteria and monitor antibiotic efficacy in vivo. In this report, we developed a novel radiopharmaceutical, 18 Ffluoromannitol ( 18 F-FMtl), which we show is specifically accumulated in both gram-positive and gram-negative bacteria but not in mammalian cells in vitro or in vivo.Methods: Clinical isolates of bacteria were serially obtained from wounds of combat service members for all in vitro and in vivo studies. The quantification of bacterial infection in vivo was performed using Positron Emission Tomography (PET)/CT imaging, and infected tissue was excised to confirm radioactivity counts ex vivo. These same tissues were used to confirm the presence of bacteria by extracting and correlating radioactive counts with colony forming units (CFU) of bacteria.Results: 18 F-FMtl was able to differentiate sterile inflammation from S. aureus and E. coli infections in vivo in a murine myositis model using PET imaging. Our study was extended to a laceration wound model infected with A. baumannii, an important pathogen in the nosocomial and battlefield setting.18 F-FMtl PET could rapidly and specifically detect infections caused by A. baumannii and several other important ESKAPE pathogens. Importantly, 18 F-FMtl PET was able to monitor therapeutic efficacy of vancomycin against S. aureus in vivo.Principal Conclusions: The ease of production of 18 F-FMtl is anticipated to facilitate wide radiopharmaceutical dissemination. Furthermore, the broad sensitivity of 18 F-FMtl for bacterial infection in vivo suggests that it is an ideal imaging agent for clinical translation to detect and monitor infections and warrants further studies in the clinical setting.
Bacillus anthracis is considered a likely agent to be used as a bioweapon, and the use of a strain resistant to the first-line antimicrobial treatments is a concern. We determined treatment efficacies against a ciprofloxacin-resistant strain of B. anthracis (Cip r Ames) in a murine inhalational anthrax model. Ten groups of 46 BALB/c mice were exposed by inhalation to 7 to 35 times the 50% lethal dose (LD 50 ) of B. anthracis Cip r Ames spores. Commencing at 36 h postexposure, groups were administered intraperitoneal doses of sterile water for injections (SWI) and ciprofloxacin alone (control groups), or ciprofloxacin combined with two antimicrobials, including meropenem-linezolid, meropenem-clindamycin, meropenem-rifampin, meropenemdoxycycline, penicillin-linezolid, penicillin-doxycycline, rifampin-linezolid, and rifampinclindamycin, at appropriate dosing intervals (6 or 12 h) for the respective antibiotics. Ten mice per group were treated for 14 days and observed until day 28. The remaining animals were euthanized every 6 to 12 h, and blood, lungs, and spleens were collected for lethal factor (LF) and/or bacterial load determinations. All combination groups showed significant survival over the SWI and ciprofloxacin controls: meropenem-linezolid (P ϭ 0.004), meropenem-clindamycin (P ϭ 0.005), meropenem-rifampin (P ϭ 0.012), meropenem-doxycycline (P ϭ 0.032), penicillindoxycycline (P ϭ 0.012), penicillin-linezolid (P ϭ 0.026), rifampin-linezolid (P ϭ 0.001), and rifampin-clindamycin (P ϭ 0.032). In controls, blood, lung, and spleen bacterial counts increased to terminal endpoints. In combination treatment groups, blood and spleen bacterial counts showed low/no colonies after 24-h treatments. The LF fell below the detection limits for all combination groups yet remained elevated in control groups. Combinations with linezolid had the greatest inhibitory effect on mean LF levels.
The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis , Burkholderia pseudomallei , B. mallei , Yersinia pestis , and Bacillus anthracis . We validated methanol treatment for B. pseudomallei . These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories.
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