skip to main content


Title: Real‐Time Monitoring of Bacteria Clearance From Blood in a Murine Model
Abstract

Bloodstream infections, especially those that are antibiotic resistant, pose a significant challenge to health care leading to increased hospitalization time and patient mortality. There are different facets to this problem that make these diseases difficult to treat, such as the difficulty to detect bacteria in the blood and the poorly understood mechanism of bacterial invasion into and out of the circulatory system. However, little progress has been made in developing techniques to study bacteria dynamics in the bloodstream. Here, we present a new approach using anin vivoflow cytometry platform for real‐time, noninvasive, label‐free, and quantitative monitoring of the lifespan of green fluorescent protein‐expressingStaphylococcus aureusandPseudomonas aeruginosain a murine model. We report a relatively fast average rate of clearance forS. aureus(k= 0.37 ± 0.09 min−1, half‐life ~1.9 min) and a slower rate forP. aeruginosa(k= 0.07 ± 0.02 min−1, half‐life ~9.6 min). We also observed what appears to be two stages of clearance forS. aureus, whileP. aeruginosaappeared only to have a single stage of clearance. Our results demonstrate that an advanced research tool can be used for studying the dynamics of bacteria cells directly in the bloodstream, providing insight into the progression of infectious diseases in circulation. © 2019 International Society for Advancement of Cytometry

 
more » « less
NSF-PAR ID:
10457034
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Cytometry Part A
Volume:
97
Issue:
7
ISSN:
1552-4922
Page Range / eLocation ID:
p. 706-712
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    We investigated competition betweenSalpa thompsoniand protistan grazers during Lagrangian experiments near the Subtropical Front in the southwest Pacific sector of the Southern Ocean. Over a month, the salp community shifted from dominance by large (> 100 mm) oozooids and small (< 20 mm) blastozooids to large (~ 60 mm) blastozooids. Phytoplankton biomass was consistently dominated by nano‐ and microphytoplankton (> 2 μm cells). Using bead‐calibrated flow‐cytometry light scatter to estimate phytoplankton size, we quantified size‐specific salp and protistan zooplankton grazing pressure. Salps were able to feed at a > 10,000 : 1 predator : prey size (linear‐dimension) ratio. Small blastozooids efficiently retained cells > 1.4μm (high end of picoplankton size, 0.6–2 μm cells) and also obtained substantial nutrition from smaller bacteria‐sized cells. Larger salps could only feed efficiently on > 5.9μm cells and were largely incapable of feeding on picoplankton. Due to the high biomass of nano‐ and microphytoplankton, however, all salps derived most of their (phytoplankton‐based) nutrition from these larger autotrophs. Phagotrophic protists were the dominant competitors for these prey items and consumed approximately 50% of the biomass of all phytoplankton size classes each day. Using a Bayesian statistical framework, we developed an allometric‐scaling equation for salp clearance rates as a function of salp and prey size:urn:x-wiley:00243590:media:lno11770:lno11770-math-0001where ESD is prey equivalent spherical diameter (µm), TL isS. thompsonitotal length,φ = 5.6 × 10−3 ± 3.6 × 10−4,ψ = 2.1 ± 0.13,θ = 0.58 ± 0.08, andγ = 0.46 ± 0.03 and clearance rate is L d‐1salp‐1. We discuss the biogeochemical and food‐web implications of competitive interactions among salps, krill, and protozoans.

     
    more » « less
  2. Abstract

    Mixotrophic nanoflagellates can account for more than half of the bacterivory in the sunlit ocean, yet very little is known about their ecophysiology. Here, we characterize the grazing ecology of an open‐ocean mixotroph in the genusFlorenciella(class Dictyochophyceae). Members of this class were indirectly implicated as major consumers ofProchlorococcusandSynechococcusin the oligotrophic North Pacific Subtropical Gyre, but their phagotrophic capabilities have never been investigated. Our studies showed thatFlorenciellareadily consumedProchlorococcus,Synechococcus, and heterotrophic bacteria, and that the ingested prey relieved nutrient limitations on growth.Florenciellagrew faster (3 d−1) in nitrogen‐deplete medium given sufficient liveSynechococcus, than in nitrogen‐replete K medium (2 d−1), but it did not grow in continuous darkness. Grazing rates were substantially higher under nutrient limitation and showed a hint of diel variability, with rates tending to be highest near the end of the light period. An apparent trade‐off between the maximum clearance rate (5 nLFlorenciella−1h−1) and the maximum ingestion rate (up to ∼ 10 prey cellsFlorenciella−1h−1) across experiments suggests that grazing behavior may also vary in response to prey concentration. If the observed grazing rates are representative of other open‐ocean mixotrophs, their collective activity could account for a significant fraction of the daily cyanobacterial mortality. This study provides essential parameters for understanding the grazing ecology of a common marine mixotroph and the first characterization of mixotrophic nanoflagellate functional responses when feeding on unicellular cyanobacteria, the dominant marine primary producers in the oligotrophic ocean.

     
    more » « less
  3. Abstract

    Not long ago, the occurrence of quantum mechanical tunneling (QMT) chemistry involving atoms heavier than hydrogen was considered unreasonable. Contributing to the shift of this paradigm, we present here the discovery of a new and distinct heavy‐atom QMT reaction. Triplet syn‐2‐formyl‐3‐fluorophenylnitrene, generated in argon matrices by UV‐irradiation of an azide precursor, was found to spontaneously cyclize to singlet 4‐fluoro‐2,1‐benzisoxazole. Monitoring the transformation by IR spectroscopy, temperature‐independent rate constants (k≈1.4×10−3 s−1; half‐life of ≈8 min) were measured from 10 to 20 K. Computational estimated rate constants are in fair agreement with experimental values, providing evidence for a mechanism involving heavy‐atom QMT through crossing triplet to singlet potential energy surfaces. Moreover, the heavy‐atom QMT takes place with considerable displacement of the oxygen atom, which establishes a new limit for the heavier atom involved in a QMT reaction in cryogenic matrices.

     
    more » « less
  4. Abstract

    Not long ago, the occurrence of quantum mechanical tunneling (QMT) chemistry involving atoms heavier than hydrogen was considered unreasonable. Contributing to the shift of this paradigm, we present here the discovery of a new and distinct heavy‐atom QMT reaction. Triplet syn‐2‐formyl‐3‐fluorophenylnitrene, generated in argon matrices by UV‐irradiation of an azide precursor, was found to spontaneously cyclize to singlet 4‐fluoro‐2,1‐benzisoxazole. Monitoring the transformation by IR spectroscopy, temperature‐independent rate constants (k≈1.4×10−3 s−1; half‐life of ≈8 min) were measured from 10 to 20 K. Computational estimated rate constants are in fair agreement with experimental values, providing evidence for a mechanism involving heavy‐atom QMT through crossing triplet to singlet potential energy surfaces. Moreover, the heavy‐atom QMT takes place with considerable displacement of the oxygen atom, which establishes a new limit for the heavier atom involved in a QMT reaction in cryogenic matrices.

     
    more » « less
  5. Introduction

    Chronic lung infection due to bacterial biofilms is one of the leading causes of mortality in cystic fibrosis (CF) patients. Among many species colonizing the lung airways,Pseudomonas aeruginosaandStaphylococcus aureusare two virulent pathogens involved in mechanically robust biofilms that are difficult to eradicate using airway clearance techniques like lung lavage. To remove such biological materials, glycoside hydrolase-based compounds are commonly employed for targeting and breaking down the biofilm matrix, and subsequently increasing cell susceptibility to antibiotics.

    Materials and methods

    In this study, we evaluate the effects of N-acetyl cysteine (NAC) and Cysteamine (CYST) in disrupting interfacial bacterial films, targeting different components of the extracellular polymeric substances (EPS). We characterize the mechanics and structural integrity of the interfacial bacterial films using pendant drop elastometry and scanning electron microscopy.

    Results and discussion

    Our results show that the film architectures are compromised by treatment with disrupting agents for 6 h, which reduces film elasticity significantly. These effects are profound in the wild type and mucoidP. aeruginosa, compared toS. aureus. We further assess the effects of competition and cooperation betweenS. aureusandP. aeruginosaon the mechanics of composite interfacial films. Films ofS. aureusand wild-typeP. aeruginosacocultures lose mechanical strength while those ofS. aureusand mucoidP. aeruginosaexhibit improved storage modulus. Treatment with NAC and CYST reduces the elastic property of both composite films, owing to the drugs’ ability to disintegrate their EPS matrix. Overall, our results provide new insights into methods for assessing the efficacy of mucolytic agents against interfacial biofilms relevant to cystic fibrosis infection.

     
    more » « less