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.

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.

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 Production and Sensing of Butyrate in a Probiotic E. coli Strain

2020 ◽  
Vol 21 (10) ◽  
pp. 3615 ◽  
Author(s):  
Yanfen Bai ◽  
Thomas J. Mansell
Keyword(s):  
Short Chain Fatty Acid ◽  
Fluorescent Protein ◽  
Coli Strain ◽  
Chain Fatty Acid ◽  
Gut Health ◽  
Human Gut ◽  
Colon Cells ◽  
E Coli ◽  
Green Fluorescent ◽  
Butyrate Production

The short-chain fatty acid butyrate plays critical roles in human gut health, affecting immunomodulation, cell differentiation, and apoptosis, while also serving as the preferred carbon source for colon cells. In this work, we have engineered a model probiotic organism, E. coli Nissle 1917 (EcN, serotype O6:K5:H1), to produce butyrate from genomic loci up to approximately 1 g/L (11 mM). Then, for real-time monitoring of butyrate production in cultures, we developed a high-throughput biosensor that responds to intracellular butyrate concentrations, with green fluorescent protein as the reporter. This work provides a foundation for studies of butyrate for therapeutic applications.

Distribution of Bacteria in Simplified Surrogate Extremities Shot With Small Caliber Projectiles

2013 ◽  
Author(s):  
Brandon J. Hinz ◽  
Karim H. Muci-Küchler ◽  
Pauline M. Smith
Keyword(s):  
High Speed ◽  
Coli Strain ◽  
Cavity Surface ◽  
Bacterial Colony ◽  
Temporary Cavity ◽  
Colony Forming Units ◽  
Biopsy Punch ◽  
K 12 ◽  
Permanent Cavity ◽  
Distribution Trends

Experiments were conducted to determine bacteria distribution trends in wound cavities of simplified surrogate extremities shot using small caliber projectiles. Two different shapes of targets, cylindrical and square, were used in this study. Cylindrical targets are more representative of an extremity but create difficulties while conducting tests due to inconsistent cavity lengths and optical distortions. Square targets, which are not as susceptible to the problems mentioned above, could be used in place of cylindrical ones if their shape does not significantly affect the distribution of bacteria within the wound cavity. Surface contamination of the targets in the experiments was represented using a circular piece of filter paper moistened with a solution with a known amount of Escherichia coli strain K-12. The projectiles used were 11.43-mm (0.45-in) caliber round nose projectiles shot from a commercially available air rifle. The permanent cavities were extracted from the targets and sliced into small, evenly spaced segments and the area surrounding the permanent cavities was removed with a biopsy punch. The radial tears that were made by the formation of the temporary cavity and surround the permanent cavity were removed using a scalpel. The permanent cavity and radial tears for each section were processed and plated on agar plates. Commercial software was used to count the number of colony forming units on each plate and the percentage of the total bacterial colony count per segment was determined. High speed video and motion analysis software was used to qualitatively and quantitatively compare the temporary cavities in the cylindrical and square targets. The data from the experiments showed that the bacteria distribution trends for the cylindrical and square targets were similar even though the maximum openings of the temporary cavity at the entrance and exit locations were higher for the cylindrical ones. For both target shapes, the bacterium was evenly distributed between the permanent cavity and the radial tears in the middle sections of the “wound tracks.” In addition, significantly higher amounts of bacterium were found in the entrance and exit segments compared with the rest of the segments in the “wound tracks”.

scholarly journals Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

2004 ◽  
Vol 70 (1) ◽  
pp. 527-534 ◽  
Author(s):  
Masahito Oda ◽  
Masatomo Morita ◽  
Hajime Unno ◽  
Yasunori Tanji
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Host Cell ◽  
Fluorescent Protein ◽  
Upstream Region ◽  
E Coli ◽  
Gene Alignment ◽  
The Stability ◽  
Green Fluorescent ◽  
K 12

ABSTRACT A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.

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