Pseudomonas Aeruginsa Research Paper
TITLE OF PROJECT: Effects of Chemoattractants of Pseudomonas Aeruginosa Through a Capillary Tube.
PROPOSED COST: $1764.90
ABSTRACT:
This project will show how strong certain bacteria that are commonly found in biofilms attract to Pseudomonas aeruginosa through a capillary tube. It will test ten bacteria to see which bacteria have more chemoattractants with Pseudomonas aeruginosa . These bacteria are all motile, so to come together to form a biofilm, they must lose their ways of mobility somehow. Since a biofilm is just one huge community of bacteria, it would be good to find out if there are ones that are more likely to come to Pseudomonas aeruginosa . It will also be done to see if there will be any difference in the ways …show more content…
that the gram positive bacteria and the gram negative bacteria attract to Pseudomonas aeruginosa . Being able to see how certain bacteria are attracted to Pseudomonas could lead to antibiotics that are good at killing certain bacteria. This could also lead to having better methods of treating certain illnesses like cystic fibrosis and even an Urinary Tract Infection.
INTRODUCTION:
Pseudomonas aeruginosa is a member of the gamma Proteobacteria class of Bacteria discovered in 1882. It is a free living bacteria commonly found in soil and water, but can also be found on surfaces of plants and animals. It is an opportunistic pathogen, meaning that is exploits some break in the host defenses to initiate an infection. There is hardly any tissue in the human body that it cannot infect (House, 2004). Pseudomonas is infamous for its resistance to antibiotics. The bacterium is naturally resistant to many antibiotics due to the permeability barrier provided by its Gram-negative outer membrane. The current classification of the genus Pseudomonas is divided into 5 groups based on ribosomal RNA (rRNA)/DNA homology. P aeruginosa is a common human saprophyte; meaning that it obtains food from dead or decaying organic matter. It rarely causes disease in healthy persons. Most infections with this organism occur in compromised hosts. Examples of compromising conditions include disrupted physical barriers to bacterial invasion, like burn injuries, IV lines, urinary catheters, dialysis catheters, endotracheal tubes, and dysfunctional immune mechanisms, such as those that appear in neonates and in individuals with cystic fibrosis. P aeruginosa is both invasive and toxigenic. The 3 stages of Pseudomonas infections are (1) bacterial attachment and colonization, (2) local infection, and (3) bloodstream dissemination and systemic disease (House, 2004) . In immunocompromised hosts, infection can progress rapidly through the 3 stages and cause pneumonia, endocarditis, peritonitis, meningitis, ecthyma gangrenosum, bacteremia, and overwhelming septicemia (House, 2004) .
Pseudomonas has a tendency to colonize surfaces in a biofilm form makes the cells impervious to therapeutic concentrations antibiotics. The typical pseudomonas might be found in a biofilm, that is attached to a substance formed like an unicellular organism (Houser, 2004). Direct observations have clearly shown that biofilm bacteria predominate, numerically and metabolically, in virtually all nutrient-sufficient ecosystems. Therefore, these sessile organisms predominate in most of the environmental, industrial, and medical problems and processes of interest to microbiologists. If biofilm bacteria were simply planktonic cells that had adhered to a surface, this revelation would be unimportant, but they are demonstrably and profoundly different. Biofilms are characterized by structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances (Horuk, 1994). Biofilms are also known for their resistance to antibiotics because the biofilm is just one huge community of bacteria, making it harder to treat an infection. Each biofilm bacterium lives in a customized microniche in a complex microbial community that has primitive homeostasis, a primitive circulatory system, and metabolic cooperativity, and each of these sessile cells reacts to its special environment so that it differs fundamentally from a planktonic cell of the same species. Biofilms can quickly undergo changes from their free-swimming, or planktonic form, to a community of bacteria. (Horuk, 1994). These cells are able to signal other bacterial cells to come together to form the biofilm. These signals are thought to be chemoattractants. The pathogenesis of P. aeruginosa is clearly multifactorial as underlined by the large number of virulence factors and the broad spectrum of diseases the bacterium causes. Many of the extracellular virulence factors required for tissue invasion and dissemination are controlled by cell-to-cell signaling systems involving homoserine lactone-based signal molecules and specific transcriptional activator proteins. These regulatory systems enable P. aeruginosa to produce virulence factors in a coordinated, cell-density–dependent manner that could allow the bacteria to overwhelm the host defense mechanisms. Interference with cell-to-cell signaling dependent virulence factor production is a promising therapeutic approach for reducing illness and death associated with P. aeruginosa colonization and infection. The growing number of human pathogens found to contain cell-to-cell signaling systems highlights the importance of exploring interference with bacterial cell-to-cell signaling for new therapeutic interventions.
Chemoattractants are inorganic or organic substances that have chemotaxis inducer effect in motile cells. In other words, chemoattractants are like a chemical agent that induces movement of chemotactic cells in the direction of its highest concentration. (Horuk, 1994). In this case, Pseudomonas would act like as a central concentration sending chemoattractants to other bacteria so that they will gather all around. The objective of this experiment would be to see which of the bacteria are more likely to come to Pseudomonas on its own without any of the other bacteria sending out chemoattractants as well.
Significance
• Help to see what Bacteria are higher in numbers in a biofilm .
• Could help in determining what antibiotics to use for an infection.
Materials & Methods
The materials that are needed for this experiment are: a Wallace 1420 Multipliable Spectrometer, Pseudomonas aeruginosa dry extract, a negative control being water, 10 motile bacteria found in biofilms, sixty test tubes, and sixty capillary tubes.
The bacteria that would need to be made into the live broth cultures are: GRAM POSITIVE--Mycobacterium tuberculosis, Corynebacterium diphtheriae, Nocardia farcinica, Rhodococcus equi, Streptomyces griseus, GRAM NEGATIVE--Escherichia coli, Salmonella typhi, Vibrio cholorae, Heliobacter pylori, and Shigella dysenteriae . Pseudomonas aeroginosa is needed to be grown on a TSA plate to get be able to get a dry extract from. The first step would be to have the dry extract of pseudomonas at the bottom of the capillary tube and then to stick the capillary tube into a test tube where there is a live broth culture of a certain bacteria at the bottom. This experiment would be repeated two more times for each of the motile …show more content…
bacteria.
MODIFIED CAPILLARY TUBE ASSAYS
The capillary tube assay developed by Pfeffer (Baker, 2006). The bacteria cells for all of the live cultures were grown in the late logarithmic phase (7.5 x 107 cells/ml) and centrifuged at 23°C for eight minutes at 1800xg and gently resuspended in a motility buffer that consists of 136.9 mM NaCl, 8.1 mM Na2HPHO4, 2.7 mM KCl, 1.47 mM KH2PO4, 2% recrystallized bovine serum albumin and .1 mM EDTA that has been adjusted to a pH of 7.4. After this is made it will be put into a test tube. The capillary tubes that have the P.aeruginosa will be placed into the test tube. After all ten bacterium have been put into the test tubes and the dry extract of Pseudomonas aeruginosa is put in the capillary tubes, it will be put into an incubator at 33°C for one hundred and twenty minutes. The capillary tubes will then be removed and the outside of the tubes need to be carefully wiped down with a paper towel. The contents will then be centrifuged at 1000 x g for three to four seconds. The bacteria will then be counted by using hemocytometry. The experiments will then be repeated two more times and the results will be in terms of the mean of the three. The standard deviation ± will also be used in the statistics. There will also be notes taken on the results of gram positive bacteria versus gram negative bacteria to see if there is any correlation between the two.
Hemocytometry
| For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to |
|determine cell concentration. A device used for determining the number of cells per unit volume of a suspension is called a counting |
|chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.|
|To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. |
|Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough|
|to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell |
|suspension. The suspension is introduced into one of the V-shaped wells a pipet. The area under the coverslip fills by capillary action. |
|Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the |
|microscope stage and the counting grid is brought into focus at low power. One entire grid on standard hemacytometers with Neubauer rulings|
|can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a |
|surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed. |
|Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly |
|distributed.
To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the |
|cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For |
|large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to |
|count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter |
|to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the |
|bottom or left ruling. |
|A way to determine a particle count using a Neubauer hemocytometer and count 187 particles in the five small squares described. Each square|
|has an area of 1/25 mm-squared (that is, 0.04 mm-squared) and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004 |
|mm-cubed. You have five squares with combined volume of 5x(0.004) = 0.02 mm-cubed. Thus you counted 187 particles in a volume of 0.02
|
|mm-cubed, giving you 187/(0.02) = 9350 particles per mm-cubed. There are 1000 cubic millimeters in one cubic centimeter (same as a |
|milliliter), so your particle count is 9,350,000 per ml. |
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BUDGET
I. Permanent Equipment:
Centrifuge
1420 Wallace Spectrometer
II. Expendable Supplies and Equipment:
Capillary Tubes $19.96
Test Tubes $79.94
TSA Plates $10.00
NaCl $20.00
Na2HPHO4 $32.00
KH2PO4 $23.00
Bovine Serum
Hemocytometer Set $226.50
iii. Bacteria Cultures
Pseudomonas aerugnosa $40.00
Mycobacterium tuberculosis $150.00
Corynebacterium diphtheria $150.00
Nocardia farcinica $150.00
Rhodococcus equi $150.00
Streptomyces griseus $150.00
Escherichia coli $150.00
Salmonella typhi $150.00
Vibrio cholorae $150.00
Heliobacter pylori $150.00
Shigella dysenteriae $150.00
=$1800.90
Literature
Dogget, R.D. Pseudomonas aeruginosa: clinical manifestations of infection and current therapy. New York. 1974
Doyle, Ron J. Biofilms. Volume 18(4) p 388 Microbial Growth. October 2002
Horuk, Richard. Chemoattractant Ligands and their Receptors. CRC Press. Boca Raton, Florida. 1996.
Jass,Jana. & Surman, Susanne. Medical Biofilms: Detection, Prevention and Control. John Wiley & Sons Publishing. New Jersey. 2003
Montie, Thomas. Biotechnology Handbooks: Pseudomonas. Vol. 10. Plenum Publishing Company. New York, New York. 1998.
Ramons, Juan- Lous. Pseudomonas: Genomics, life style and molecular architecture. Springer Publishing. 2007.
Thrall,Anna and Dale C. Baker. Veterinary Hematology and Clinical Chemistry: Text and Clinical Case Presentations Set. New York, New York 2004.
PROPOSED COST: $1764.90
ABSTRACT:
This project will show how strong certain bacteria that are commonly found in biofilms attract to Pseudomonas aeruginosa through a capillary tube. It will test ten bacteria to see which bacteria have more chemoattractants with Pseudomonas aeruginosa . These bacteria are all motile, so to come together to form a biofilm, they must lose their ways of mobility somehow. Since a biofilm is just one huge community of bacteria, it would be good to find out if there are ones that are more likely to come to Pseudomonas aeruginosa . It will also be done to see if there will be any difference in the ways …show more content…
that the gram positive bacteria and the gram negative bacteria attract to Pseudomonas aeruginosa . Being able to see how certain bacteria are attracted to Pseudomonas could lead to antibiotics that are good at killing certain bacteria. This could also lead to having better methods of treating certain illnesses like cystic fibrosis and even an Urinary Tract Infection.
INTRODUCTION:
Pseudomonas aeruginosa is a member of the gamma Proteobacteria class of Bacteria discovered in 1882. It is a free living bacteria commonly found in soil and water, but can also be found on surfaces of plants and animals. It is an opportunistic pathogen, meaning that is exploits some break in the host defenses to initiate an infection. There is hardly any tissue in the human body that it cannot infect (House, 2004). Pseudomonas is infamous for its resistance to antibiotics. The bacterium is naturally resistant to many antibiotics due to the permeability barrier provided by its Gram-negative outer membrane. The current classification of the genus Pseudomonas is divided into 5 groups based on ribosomal RNA (rRNA)/DNA homology. P aeruginosa is a common human saprophyte; meaning that it obtains food from dead or decaying organic matter. It rarely causes disease in healthy persons. Most infections with this organism occur in compromised hosts. Examples of compromising conditions include disrupted physical barriers to bacterial invasion, like burn injuries, IV lines, urinary catheters, dialysis catheters, endotracheal tubes, and dysfunctional immune mechanisms, such as those that appear in neonates and in individuals with cystic fibrosis. P aeruginosa is both invasive and toxigenic. The 3 stages of Pseudomonas infections are (1) bacterial attachment and colonization, (2) local infection, and (3) bloodstream dissemination and systemic disease (House, 2004) . In immunocompromised hosts, infection can progress rapidly through the 3 stages and cause pneumonia, endocarditis, peritonitis, meningitis, ecthyma gangrenosum, bacteremia, and overwhelming septicemia (House, 2004) .
Pseudomonas has a tendency to colonize surfaces in a biofilm form makes the cells impervious to therapeutic concentrations antibiotics. The typical pseudomonas might be found in a biofilm, that is attached to a substance formed like an unicellular organism (Houser, 2004). Direct observations have clearly shown that biofilm bacteria predominate, numerically and metabolically, in virtually all nutrient-sufficient ecosystems. Therefore, these sessile organisms predominate in most of the environmental, industrial, and medical problems and processes of interest to microbiologists. If biofilm bacteria were simply planktonic cells that had adhered to a surface, this revelation would be unimportant, but they are demonstrably and profoundly different. Biofilms are characterized by structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances (Horuk, 1994). Biofilms are also known for their resistance to antibiotics because the biofilm is just one huge community of bacteria, making it harder to treat an infection. Each biofilm bacterium lives in a customized microniche in a complex microbial community that has primitive homeostasis, a primitive circulatory system, and metabolic cooperativity, and each of these sessile cells reacts to its special environment so that it differs fundamentally from a planktonic cell of the same species. Biofilms can quickly undergo changes from their free-swimming, or planktonic form, to a community of bacteria. (Horuk, 1994). These cells are able to signal other bacterial cells to come together to form the biofilm. These signals are thought to be chemoattractants. The pathogenesis of P. aeruginosa is clearly multifactorial as underlined by the large number of virulence factors and the broad spectrum of diseases the bacterium causes. Many of the extracellular virulence factors required for tissue invasion and dissemination are controlled by cell-to-cell signaling systems involving homoserine lactone-based signal molecules and specific transcriptional activator proteins. These regulatory systems enable P. aeruginosa to produce virulence factors in a coordinated, cell-density–dependent manner that could allow the bacteria to overwhelm the host defense mechanisms. Interference with cell-to-cell signaling dependent virulence factor production is a promising therapeutic approach for reducing illness and death associated with P. aeruginosa colonization and infection. The growing number of human pathogens found to contain cell-to-cell signaling systems highlights the importance of exploring interference with bacterial cell-to-cell signaling for new therapeutic interventions.
Chemoattractants are inorganic or organic substances that have chemotaxis inducer effect in motile cells. In other words, chemoattractants are like a chemical agent that induces movement of chemotactic cells in the direction of its highest concentration. (Horuk, 1994). In this case, Pseudomonas would act like as a central concentration sending chemoattractants to other bacteria so that they will gather all around. The objective of this experiment would be to see which of the bacteria are more likely to come to Pseudomonas on its own without any of the other bacteria sending out chemoattractants as well.
Significance
• Help to see what Bacteria are higher in numbers in a biofilm .
• Could help in determining what antibiotics to use for an infection.
Materials & Methods
The materials that are needed for this experiment are: a Wallace 1420 Multipliable Spectrometer, Pseudomonas aeruginosa dry extract, a negative control being water, 10 motile bacteria found in biofilms, sixty test tubes, and sixty capillary tubes.
The bacteria that would need to be made into the live broth cultures are: GRAM POSITIVE--Mycobacterium tuberculosis, Corynebacterium diphtheriae, Nocardia farcinica, Rhodococcus equi, Streptomyces griseus, GRAM NEGATIVE--Escherichia coli, Salmonella typhi, Vibrio cholorae, Heliobacter pylori, and Shigella dysenteriae . Pseudomonas aeroginosa is needed to be grown on a TSA plate to get be able to get a dry extract from. The first step would be to have the dry extract of pseudomonas at the bottom of the capillary tube and then to stick the capillary tube into a test tube where there is a live broth culture of a certain bacteria at the bottom. This experiment would be repeated two more times for each of the motile …show more content…
bacteria.
MODIFIED CAPILLARY TUBE ASSAYS
The capillary tube assay developed by Pfeffer (Baker, 2006). The bacteria cells for all of the live cultures were grown in the late logarithmic phase (7.5 x 107 cells/ml) and centrifuged at 23°C for eight minutes at 1800xg and gently resuspended in a motility buffer that consists of 136.9 mM NaCl, 8.1 mM Na2HPHO4, 2.7 mM KCl, 1.47 mM KH2PO4, 2% recrystallized bovine serum albumin and .1 mM EDTA that has been adjusted to a pH of 7.4. After this is made it will be put into a test tube. The capillary tubes that have the P.aeruginosa will be placed into the test tube. After all ten bacterium have been put into the test tubes and the dry extract of Pseudomonas aeruginosa is put in the capillary tubes, it will be put into an incubator at 33°C for one hundred and twenty minutes. The capillary tubes will then be removed and the outside of the tubes need to be carefully wiped down with a paper towel. The contents will then be centrifuged at 1000 x g for three to four seconds. The bacteria will then be counted by using hemocytometry. The experiments will then be repeated two more times and the results will be in terms of the mean of the three. The standard deviation ± will also be used in the statistics. There will also be notes taken on the results of gram positive bacteria versus gram negative bacteria to see if there is any correlation between the two.
Hemocytometry
| For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to |
|determine cell concentration. A device used for determining the number of cells per unit volume of a suspension is called a counting |
|chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.|
|To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. |
|Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough|
|to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell |
|suspension. The suspension is introduced into one of the V-shaped wells a pipet. The area under the coverslip fills by capillary action. |
|Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the |
|microscope stage and the counting grid is brought into focus at low power. One entire grid on standard hemacytometers with Neubauer rulings|
|can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a |
|surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed. |
|Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly |
|distributed.
To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the |
|cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For |
|large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to |
|count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter |
|to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the |
|bottom or left ruling. |
|A way to determine a particle count using a Neubauer hemocytometer and count 187 particles in the five small squares described. Each square|
|has an area of 1/25 mm-squared (that is, 0.04 mm-squared) and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004 |
|mm-cubed. You have five squares with combined volume of 5x(0.004) = 0.02 mm-cubed. Thus you counted 187 particles in a volume of 0.02
|
|mm-cubed, giving you 187/(0.02) = 9350 particles per mm-cubed. There are 1000 cubic millimeters in one cubic centimeter (same as a |
|milliliter), so your particle count is 9,350,000 per ml. |
| |
| |
| |
| |
| |
| |
| |
| |
| |
BUDGET
I. Permanent Equipment:
Centrifuge
1420 Wallace Spectrometer
II. Expendable Supplies and Equipment:
Capillary Tubes $19.96
Test Tubes $79.94
TSA Plates $10.00
NaCl $20.00
Na2HPHO4 $32.00
KH2PO4 $23.00
Bovine Serum
Hemocytometer Set $226.50
iii. Bacteria Cultures
Pseudomonas aerugnosa $40.00
Mycobacterium tuberculosis $150.00
Corynebacterium diphtheria $150.00
Nocardia farcinica $150.00
Rhodococcus equi $150.00
Streptomyces griseus $150.00
Escherichia coli $150.00
Salmonella typhi $150.00
Vibrio cholorae $150.00
Heliobacter pylori $150.00
Shigella dysenteriae $150.00
=$1800.90
Literature
Dogget, R.D. Pseudomonas aeruginosa: clinical manifestations of infection and current therapy. New York. 1974
Doyle, Ron J. Biofilms. Volume 18(4) p 388 Microbial Growth. October 2002
Horuk, Richard. Chemoattractant Ligands and their Receptors. CRC Press. Boca Raton, Florida. 1996.
Jass,Jana. & Surman, Susanne. Medical Biofilms: Detection, Prevention and Control. John Wiley & Sons Publishing. New Jersey. 2003
Montie, Thomas. Biotechnology Handbooks: Pseudomonas. Vol. 10. Plenum Publishing Company. New York, New York. 1998.
Ramons, Juan- Lous. Pseudomonas: Genomics, life style and molecular architecture. Springer Publishing. 2007.
Thrall,Anna and Dale C. Baker. Veterinary Hematology and Clinical Chemistry: Text and Clinical Case Presentations Set. New York, New York 2004.