Effect of Initial Surface Concentration on Bacterial Distribution in a Surrogate Ballistic Wound

2011 ◽  
Author(s):  
Meaghen A. Krebsbach ◽  
Karim H. Muci-Ku¨chler
Keyword(s):  
Fluorescent Protein ◽  
Surface Concentration ◽  
Skin Surface ◽  
Coli Strain ◽  
Initial Surface ◽  
Bacterial Concentration ◽  
Bacterial Colony ◽  
Initial Surface Concentration ◽  
Green Fluorescent ◽  
K 12

In ballistic injuries, contamination can be carried from the environment, clothing, and skin surface into the wound track. Bacteria and contaminated debris can be introduced into the wound by several means, including physical transport by the projectile or by the suction caused by the formation and collapse of the temporary wound cavity. In this paper, the relationship between initial bacterial concentration on the surface and resultant bacterial distribution along the wound channel is examined using a leg surrogate. Escherichia coli strain K-12 was used to represent skin surface contamination. In order to reduce the possibility of contamination by outside bacteria and assist in colony visualization, the E. coli first underwent a transformation protocol to express Green Fluorescent Protein and to be resistant to the antibiotic ampicillin. Different concentrations of bacteria were pipetted onto circular filter paper and placed onto the surface of a ballistic gelatin leg surrogate, and an 11.43-mm (0.45-in) caliber projectile was shot through the contaminated area into the gel. The “wound track” was sliced into small, evenly spaced samples and the permanent cavity was removed using a biopsy punch, liquefied, and grown on selective lysogeny broth media containing ampicillin. Examination of a normalized bacterial colony count and normalized area covered per segment allowed comparison of variations in the initial concentration, and confirmed that within a range the normalized contamination distribution trend along the “wound track” remained similar. This verification allowed additional confidence in results obtained using this bacteria distribution methodology by eliminating concerns over small variations in initial bacterial concentration.

Influence of Projectile Momentum and Kinetic Energy on Bacterial Distribution in an Extremity Surrogate “Wound Track”

2012 ◽  
Author(s):  
Meaghen A. Krebsbach ◽  
Karim H. Muci-Küchler ◽  
Brandon J. Hinz
Keyword(s):  
Kinetic Energy ◽  
Filter Paper ◽  
Fluorescent Protein ◽  
Coli Strain ◽  
Initial Kinetic Energy ◽  
Initial Surface ◽  
Bacterial Colony ◽  
Initial Momentum ◽  
Green Fluorescent ◽  
K 12

This paper presents an experimental study that examines the relationship between the initial momentum and the initial kinetic energy of a projectile and the distribution of bacterial contamination along a “wound track” created in an extremity surrogate representative of the superior (upper) portion of the lower leg (i.e., the calf region) of an average adult human male. Initial surface contamination was represented using circular filter paper moistened with a solution containing 5 × 106 colony forming units per milliliter (CFU/ml) of Escherichia coli strain K-12 that was previously transformed to express green fluorescent protein (GFP) and be resistant to ampicillin. The contaminated filter paper and extremity surrogate were perforated with 11.43-mm (0.45-in) caliber round nose lead projectiles shot from commercially available air rifles. To match the initial momentum and/or kinetic energy between experiments, 11.0 g (170 grain) and 14.9 g (230 grain) projectiles were shot at velocities ranging from 145 m/s to 195 m/s. The “wound track” was extracted from the extremity surrogate and sliced into small, evenly spaced segments and the permanent cavity was removed from each segment using a biopsy punch, liquefied, and grown on selective agar containing ampicillin. Examination of the bacterial colony count and area covered by bacteria colonies per segment allowed comparison of differences between trends in the bacteria distribution along the “wound track”. The results obtained showed that, for the cases considered, the bacterial distribution trends were similar for the experimental groups with like initial kinetic energies.

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

Survival of Salmonella on Tomatoes Stored at High Relative Humidity, in Soil, and on Tomatoes in Contact with Soil

2002 ◽  
Vol 65 (2) ◽  
pp. 274-279 ◽  
Author(s):  
XUAN GUO ◽  
JINRU CHEN ◽  
ROBERT E. BRACKETT ◽  
LARRY R. BEUCHAT
Keyword(s):  
Relative Humidity ◽  
Fruits And Vegetables ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Skin Surface ◽  
Contaminated Water ◽  
Protein Marker ◽  
Green Fluorescent Protein Marker ◽  
Salmonella Serotypes ◽  
Green Fluorescent

Salmonellosis has been linked to the consumption of several types of raw fruits and vegetables, some of which may have been contaminated with Salmonella before harvesting. The objectives of this study were to investigate water and soil as reservoirs of Salmonella for the contamination of mature green tomato fruits. Salmonella survived for at least 45 days in inoculated moist soil. The population of Salmonella on tomatoes in contact with soil increased by 2.5 log10 CFU per tomato during storage for 4 days at 20°C and remained constant for an additional 10 days. The number of cells inoculated on tomatoes decreased by approximately 4 log10 CFU per tomato during storage for 14 days at 20°C and 70% relative humidity. Fruits in contact with inoculated soil for 1 day at 20°C harbored Salmonella only near or on the skin surface. More Salmonella cells were observed in stem scar and subsurface areas of tomatoes as the time of storage increased. PCR fingerprinting revealed that among five Salmonella serotypes in the inoculum, Salmonella Montevideo was the most persistent on tomatoes in contact with inoculated soil and on spot-inoculated tomatoes, followed by Salmonella Poona and Salmonella Michigan. The results of this study demonstrate that an enhanced green fluorescent protein marker can be used to detect cells and monitor the growth of Salmonella in the presence of other microorganisms. Observations on the infiltration of Salmonella into tomato tissues support the contention that preharvest contact of produce with contaminated water or soil exacerbates problems associated with the postharvest removal of pathogens or their accessibility to treatment with sanitizers.

Effect of Projectile Caliber and Speed on Bacterial Distribution in a Leg Surrogate

2010 ◽  
Author(s):  
Meaghen A. Krebsbach ◽  
Karim H. Muci-Ku¨chler ◽  
Brandon J. Hinz
Keyword(s):  
Filter Paper ◽  
Fluorescent Protein ◽  
Contamination Level ◽  
Bacterial Colony ◽  
E Coli ◽  
Biopsy Punch ◽  
Ballistic Gelatin ◽  
Green Fluorescent ◽  
The Relationship ◽  
Permanent Cavity

This paper examines the relationship between ballistic factors and bacterial distribution along a surrogate wound channel using ballistic gelatin cylinders with dimensions representative of the calf region of an average human leg. The ballistics factors considered were projectile caliber and speed, and Escherichia coli (E. coli) was used as the representative bacteria. In order to reduce the possibility of contamination by outside bacteria, the E. coli first underwent a transformation protocol to express Green Fluorescent Protein (GFP) and become resistant to the antibiotic ampicillin. A set volume of bacteria was pipetted onto a small piece of filter paper which was placed on the surface of a ballistic gelatin cylinder and a projectile was shot through the bacteria saturated filter paper. The ‘wound track’ was divided into slices, and the area surrounding the permanent cavity was removed with a biopsy punch, liquefied, and grown on selective LB media containing ampicillin. Examination of the bacterial colony count along the permanent cavity segments allowed comparison of how variations in projectile caliber and speed affected contamination distribution along the ‘wound track’. Initial results indicate that larger calibers may result in higher contamination distribution at the projectile entrance and exit regions and higher speeds compress the distribution and result in a drop in contamination level near the exit.

Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo

2005 ◽  
Vol 61 (3) ◽  
pp. 389-398 ◽  
Author(s):  
Michael Schultz ◽  
Sonja Watzl ◽  
Tobias A. Oelschlaeger ◽  
Heiko C. Rath ◽  
Claudia Göttl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Coli Strain ◽  
Green Fluorescent ◽  
Probiotic Microorganism

Transformation of Escherichia coli K-12 with a High-Copy Plasmid Encoding the Green Fluorescent Protein Reduces Growth: Implications for Predictive Microbiology†

2006 ◽  
Vol 69 (2) ◽  
pp. 276-281 ◽  
Author(s):  
T. P. OSCAR ◽  
K. DULAL ◽  
D. BOUCAUD
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Predictive Models ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
E Coli ◽  
Contaminated Food ◽  
Green Fluorescent ◽  
K 12 ◽  
High Copy Plasmid

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (μmax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40°C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40°C, μmax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

scholarly journals Location and dynamics of an active promoter in Escherichia coli K-12

2011 ◽  
Vol 441 (1) ◽  
pp. 481-485 ◽  
Author(s):  
María-Antonia Sánchez-Romero ◽  
David J. Lee ◽  
Eugenio Sánchez-Morán ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Lac Repressor ◽  
Fluorescence Signal ◽  
Bacterial Cells ◽  
Protein Fusion ◽  
Lac Operator ◽  
Low Copy Number ◽  
Green Fluorescent ◽  
K 12

In the present paper, we report that transcription affects the location of a DNA target in Escherichia coli K-12. A strain whose chromosome had been engineered to encode a lac repressor–GFP (green fluorescent protein) fusion was used as a host for a low copy number plasmid that carries an array of five lac operator sites. Individual cells of this strain exhibited a diffuse fluorescence signal, suggesting that the plasmid is distributed throughout the cell cytoplasm. However, a derivative of this plasmid carrying a cloned constitutive promoter is targeted to a location at the edge of the nucleoid towards the pole of the host cell. We conclude that transcription from the cloned promoter is driving the location of the plasmid and that specific locations in bacterial cells may favour gene expression.

scholarly journals mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

2013 ◽  
Vol 79 (20) ◽  
pp. 6362-6368 ◽  
Author(s):  
Ying Xu ◽  
Bing Chen ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Transport Activity ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent ◽  
K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

scholarly journals Determination of the time-dependent change in the vapor concentration of 2,4,6-trinitrotoluene during the sublimation of its traces from the glass surface

2021 ◽  
Vol 25 (3) ◽  
pp. 222-229
Author(s):  
M. I. Tivileva ◽  
◽  
V. M. Gruznov ◽  
M. N. Baldin ◽  
A. V. Kikhtenko ◽  
...  
Keyword(s):  
Glass Surface ◽  
Steel Wire ◽  
Surface Concentration ◽  
Threshold Concentration ◽  
Wire Mesh ◽  
Vapor Concentration ◽  
Initial Surface ◽  
Dependent Change ◽  
Initial Surface Concentration ◽  
Steel Wire Mesh

The results of the measurements of 2,4,6-trinitrotoluene (TNT) vapor concentration over its trace amounts, called thin films, on the glass surface with a concentration of 100 ng/cm2 in a square area with a side of 1 cm over time are presented. The trace amounts of TNT on the glass were formed by applying a solution of TNT in the acetonitrile diluted with the chemically pure acetone, followed by the evaporation of the solvents. In order to measure the TNT vapor concentration, an EKHO-V-IDTS portable multibacillary gas-chromatograph with preliminary TNT vapor concentration was used. A sampling of the TNT vapor above the object was carried out with a remote vortex sampler. The vapor sample was taken from a distance of 2 cm from the glass surface. The concentration in the mode of the complete capture of TNT vapors was carried out to the stainless-steel wire mesh. The vapor concentration was determined from the chromatographic peak amplitude. It was found that the concentration of vapor over the examined surface with an area of 1 cm2 decreases from 10-13 to 10-14 g/cm3 within 2.6 ± 0.3 hours. TNT vapor concentration value of 10-14 g/cm3 corresponds to the threshold concentration of TNT vapor for the modern detectors. Based on the assumption that the vapor concentration is proportional to the amount of the TNT mass on the surface for the considered trace amounts of TNT, it was estimated that the initial surface concentration of trinitrotoluene of 100 ng/cm2 on the glass surface decreases to 12 ng/cm2 within 2.6 ± 0.3 hours due to sublimation into an open half-space. It was shown that the use of vortex sampling of vapor intensifies the sublimation of TNT from the glass surface.

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