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Design of a Tracheal Model for Testing Endotracheal Tube Bacteria Adhesion

Permanent Link: http://ufdc.ufl.edu/UFE0022106/00001

Material Information

Title: Design of a Tracheal Model for Testing Endotracheal Tube Bacteria Adhesion
Physical Description: 1 online resource (103 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: adhesion, bacteria, biofilm, endotracheal, intubation, mucus, pneumonia, trachea
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
Genre: Aerospace Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Every day millions of patients are intubated. The insertion of an endotracheal tube in the trachea, the stomach, or the nasal tract can lead to several complications. Bacteria adhere to most of the implanted medical devices or damaged tissues and in the case of a patient affected by a lung disease, intubation usually leads to bacteria colonization of main airways. Our objective is to better understand bacterial adhesion of the tracheal tract and the endotracheal tubes. For that purpose, we have built a flow system device to simulate the human tracheal environment, composed of mucus and air flows. The aim of this cylindrical flow chamber is to test endotracheal tubes in a diseased environment. Natural tracheal mucus is used to create a model closer to the reality. An experimental design has also been developed for assessing the role and influence of various parameters on the adhesion of bacteria on endotracheal tubes under physiological conditions. This new experimental setup could lead not only to a better understanding of bacteria adhesion, but also to the design of novel and more efficient endotracheal tubes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Tran-Son-Tay, Roger.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022106:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022106/00001

Material Information

Title: Design of a Tracheal Model for Testing Endotracheal Tube Bacteria Adhesion
Physical Description: 1 online resource (103 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: adhesion, bacteria, biofilm, endotracheal, intubation, mucus, pneumonia, trachea
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
Genre: Aerospace Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Every day millions of patients are intubated. The insertion of an endotracheal tube in the trachea, the stomach, or the nasal tract can lead to several complications. Bacteria adhere to most of the implanted medical devices or damaged tissues and in the case of a patient affected by a lung disease, intubation usually leads to bacteria colonization of main airways. Our objective is to better understand bacterial adhesion of the tracheal tract and the endotracheal tubes. For that purpose, we have built a flow system device to simulate the human tracheal environment, composed of mucus and air flows. The aim of this cylindrical flow chamber is to test endotracheal tubes in a diseased environment. Natural tracheal mucus is used to create a model closer to the reality. An experimental design has also been developed for assessing the role and influence of various parameters on the adhesion of bacteria on endotracheal tubes under physiological conditions. This new experimental setup could lead not only to a better understanding of bacteria adhesion, but also to the design of novel and more efficient endotracheal tubes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Tran-Son-Tay, Roger.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022106:00001


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1 DESIGN OF A TRACHEAL MODEL FOR TESTING ENDOTRACHEAL TUBE BACTERIA ADHESION By KARINE VIZCAINO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Karine Vizcaino

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3 To Claude and Dominique Vizcaino for their never-ending support.

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4 ACKNOWLEDGMENTS I acknowled ge my supervisory committee for providing the guidance necessary to complete my masters degree. I especially thank Dr. Tran-Son-Tay (Chair), Dr. Antonelli, Dr. Sarntinoranont and Edith Angel Sampson for their help. I thank my parents, Claude and Dominique Vizc aino, for their endless support and belief in my project. I thank my mom for being patie nt, always thinking I would succeed in my international projects and supporting me in all the hard steps I went through. I give special thanks to my best friend Christian Paccard for supporting me and understanding me as well as he did during this long period abroad.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................12 CHAP TER 1 INTRODUCTION..................................................................................................................13 2 OBJECTIVES.........................................................................................................................15 Introduction................................................................................................................... ..........15 Rationale.................................................................................................................................15 Objectives...............................................................................................................................16 3 BACKGROUND.................................................................................................................... 17 Trachea Description............................................................................................................ ....17 Mucus Description..................................................................................................................17 Lung Diseases, Intubation and Effects on the Tracheal Environm ent and the Secretions..... 19 Bacteria and Adhesion............................................................................................................23 Surface Chemical Compositions Influencing Adhesion..................................................24 Bacterial Properties Influencing Adhesion...................................................................... 25 Biofilms..................................................................................................................................25 Biofilm Formation........................................................................................................... 26 Forces Governing the First Step of Adhesion................................................................. 27 Interaction and Complementarities between Bacteria ..................................................... 27 Pseudomonas Biofilm s and Antibiotics Resistance............................................................... 28 Adhesion Tests........................................................................................................................28 Static Adherence Method................................................................................................ 28 Dynamic Method.............................................................................................................30 Comparison of the Static and Dynamic Methods............................................................ 30 Tracheal Models................................................................................................................ .....31 4 DESIGN OF A TRACHEAL MODEL.................................................................................. 34 Specifications..........................................................................................................................34 Design of the Tracheal Model and Method............................................................................ 35 General Principle of Functioning....................................................................................35 Calculations of the Dimensions....................................................................................... 37

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6 Description of the Different Parts ...........................................................................................39 Base.................................................................................................................................39 Cylindrical Bottom.......................................................................................................... 40 Mucus Tank.....................................................................................................................40 Mucus and Air Tightness of the Device................................................................................. 43 Modifications..........................................................................................................................44 5 CHARACTERIZATION OF THE FLOW............................................................................. 47 Materials.................................................................................................................................47 Methods..................................................................................................................................48 Results.....................................................................................................................................48 Dextran Solution..............................................................................................................48 Homogeneity and Characterization of the Flow.............................................................. 49 6 BACTERIA ADHESION TEST............................................................................................53 Materials.................................................................................................................................53 Methods..................................................................................................................................55 Bacteria Count........................................................................................................................55 Results and Discussion......................................................................................................... ..56 7 FUTURE WORK AND CONCLUSION............................................................................... 60 Type of Experiments............................................................................................................ ...60 Topographies of the Endotracheal Tube.................................................................................60 Conclusion..............................................................................................................................62 APPENDIX A INTUBATION, DISEASED EN VIRONME NT AND BACTERIA..................................... 63 Pseudomonas Aeruginosa .......................................................................................................63 Intubation................................................................................................................................64 B FLOW CHAMBER DESIGN................................................................................................. 65 Mucus Flow............................................................................................................................66 Reynolds Number................................................................................................................ ...66 Calculus of the Mucus Flow Characteristics.......................................................................... 67 Size a Parallel-Plate Flow Chamber.......................................................................................68 Similitudes.................................................................................................................... ..........68 C JOURNAL OF DRAWINGS................................................................................................. 70 D FLOW CHARACTERIZATION............................................................................................83

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7 E EXPERIMENTAL PROTOCOL FOR BACTERIA ADHESION TESTS ............................86 Objective.................................................................................................................................86 Material List............................................................................................................................86 Disassembly of the Tracheal Flow Chamber.......................................................................... 86 Autoclave Procedure............................................................................................................ ...90 Material Preparation........................................................................................................90 Loading Autoclave..........................................................................................................90 Operating Autoclave........................................................................................................ 90 Unloading Autoclave....................................................................................................... 90 Sterilization and Preparation of the Material ..........................................................................91 Assembly of the Tracheal Flow Chamber under the Biological Hood ................................... 92 Egg Whites Preparation......................................................................................................... .92 Culture Preparation.................................................................................................................93 Coating Method......................................................................................................................93 Operating Procedure...............................................................................................................94 Bacteria Count........................................................................................................................95 LIST OF REFERENCES...............................................................................................................98 BIOGRAPHICAL SKETCH.......................................................................................................103

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8 LIST OF FIGURES Figure page 3-1 Prevalence of VAP related to the numb er of days on m echanical ventilation................... 21 3-2 Biofilm formation and growth........................................................................................... 27 3-3 Horizontal tracheal model.................................................................................................. 31 4-1 Schematic of the diseased patient tracheal model.............................................................. 35 4-2 Tracheal model............................................................................................................. ......37 4-3 Mucus reservoir geometry................................................................................................. 38 4-4 Wall shear stress.......................................................................................................... ......38 4-5 Shear stress on the mucus layer......................................................................................... 39 4-6 Cylindrical bottom......................................................................................................... ....40 4-7 Mucus tank and top cover.................................................................................................. 41 4-8 Exploded view of the device.............................................................................................. 42 4-9 O-rings position........................................................................................................... ......43 4-10 Adjustable endotracheal fitting.......................................................................................... 44 5-1 Brookfield viscometer...................................................................................................... ..47 5-2 Dextran solution........................................................................................................... ......48 5-3 Mean dynamic viscosity of 25% dextran solution.............................................................49 5-4 Experiment showing th e beginning of the flow................................................................. 50 5-5 Flow around the cuff tube..................................................................................................51 5-6 Mucus reservoir flow....................................................................................................... ..51 5-7 Aspect of the coating for different flow rates.................................................................... 52 6-1 Experimental set-up........................................................................................................ ...54 6-2 Dried egg white in th e tracheal flow cham ber................................................................... 56 6-3 Agar plates with P. aeruginosa isolated from the egg white contained in the syringe...... 57

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9 6-4 Aspect of the biofilm surfactant solution containing ETT segments................................. 58 6-5 Agar plate showing the bacteria growth for each ETT segment........................................ 58 7-1 Micro-topographies fabricated in PDMSe from silicon wafer templates.......................... 61 A-1 Schematic of bacteria.........................................................................................................63 A-2 Pseudomonas aeruginosa infection of hum an respirator y mucosa in an organ culture caused patchy epithelial damage after 8 h. P. aeruginosa adhered to mucus and damaged epithelium, but not to normal epithelium........................................................... 63 A-3 Endotracheal tube after being placed through the vocal cords and keeping them open.... 64 B-1 Design of a parallel-plate flow chamber............................................................................65 B-2 Cylinder-shaped model of the trachea...............................................................................66 B-3 Dimensions of the flow chamber (l0, w0 and h0) and distances in the different directions (l, w, h)........................................................................................................... ...67 D-1 Initial flow: Time 0:00 to 5:30........................................................................................... 83 D-2 Flow evolution: Time 7:00 to 14:00.................................................................................. 84 D-3 Final flow covering the entire surface of the lexan tube: Tim e 15:00 to 25:00................. 85 E-1 Tubings and connector, mucus entrance............................................................................ 86 E-2 Tracheal flow chamber and the corresponding part numbers............................................ 87 E-3 Parts 1, 2, 3, 4 disassembled.............................................................................................. 88 E-4 Mucus reservoir disassembled (parts 9 and 11).................................................................88 E-5 Feet disassembled.......................................................................................................... ....88 E-6 Cylindrical bottom (part 10) and fitting balloon (part 13) ................................................. 89 E-7 Device disassembled........................................................................................................ ..89 E-8 Autoclave parts............................................................................................................ ......91 E-9 Endotracheal tube segments marks.................................................................................... 92 E-10 Egg white separation..........................................................................................................93 E-11 Coating operation......................................................................................................... ......94

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10 E-12 Experimental set up....................................................................................................... .....95 E-13 Extraction of the endotracheal tube................................................................................... 96 E-14 Cutting method............................................................................................................ .......96 E-15 The standard plate count technique to de term ine the total number of microorganisms in a sample.................................................................................................................... .....97

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11 LIST OF ABBREVIATIONS AFM Atomic force microscopy CF Cystic fibrosis CFU Colony forming units ETT Endotracheal tube ICU Intensive care units P. aeruginosa Pseudomonas aeruginosa PBS Phosphate buffered saline PDMSe Polydimethylsiloxane elastomer PTFE Teflon SEM Scanning electron microscopy TSB Triptic soy broth VAP Ventilator-associated pneumonia

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12 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DESIGN OF A TRACHEAL MODEL FOR TE STING ENDOTRACHEAL TUBE BACTERIA ADHESION By Karine Vizcaino May 2008 Chair: Roger Tran-Son-Tay Major: Aerospace Engineering Every day millions of patients are intubated. The insertion of an endotracheal tube in the trachea, the stomach, or the nasal tract can lead to several complications. Ba cteria adhere to most of the implanted medical devices or damaged tissu es and in the case of a patient affected by a lung disease, intubation usually lead s to bacteria colonization of ma in airways. Our objective is to better understand bacterial adhe sion of the tracheal tract and the endotracheal tubes. For that purpose, we have built a flow system device to simulate the human tracheal environment, composed of mucus and air flows. The aim of this cylindrical flow chamber is to test endotracheal tubes in a diseased environment. Na tural tracheal mucus is used to create a model closer to the reality. An experi mental design has also been deve loped for assessing the role and influence of various parameters on the adhesi on of bacteria on e ndotracheal tubes under physiological conditions. This new experiment al setup could lead not only to a better understanding of bacteria adhesion, but also to the design of novel and more efficient endotracheal tubes.

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13 CHAPTER 1 INTRODUCTION Approxim ately 25.8 million disposable endotracheal tubes (ETT) were sold in the UnitedStates in 2002 (source: Frost and Sullivan 2003). Intubations are require d not only for patients admitted to Intensive Cares Units (ICU) but al so in any kind of operations that require anesthesia. In the case of patients affected by lungs diseases such as cystic Fibrosis or Pneumonia, the intubation is required for several months. Therefor e, the use of the endotra cheal tubes represents a main risk factor for those immuno-compromised patients. The ETT is inserted down the trachea; bacteria colonize, adhere to its su rface and can freely move into the body. A patient affected by pneumonia can develop ventilator-a ssociated pneumonia (VAP) after only 48 hours. Pseudomonas is an opportunistic organism that adhe res to most of the foreign body surface and injured tissues. It is a bacterium hard to eradicat e as it grows in biofilms, and one in ten hospitalacquired infections are from them. Twenty-eig ht percent of patients receiving mechanical ventilation will continue to have complication of VAP. The mortality ranges are from 28 to 70% (Rumbak 2002). VAP cost approximately $40,00 0 per case, and there are 300,000 new cases annually in the United States. It is important to work on a treatment of this pathology and to prevent severe infections and contaminations. The length of stay on mechanical ventilation increases the colonization of the upper airways and this is the reason why intubation is considered as a major risk factor. The ventilator tubing is an important source of contamination. As Pseudomonas aeruginosa are hard to eradicate by antibiotic mean, a way to reduce those infections would be to minimize the bacteria l adhesion on the endotracheal tube. To minimize bacteria adhesion on those tubes it is important to identify and control the key parameters affecting bacteria adhesion. Becau se of the complexity of th e problem, a tracheal model of

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14 diseased patients is needed in order to assess the role of various factors, like temperature, humidity, mucus properties etc. This knowledge will lead to a better understanding of the adhesion process.

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15 CHAPTER 2 OBJECTIVES Introduction Understanding bacteria adhesion in a diseased tracheal env ironmen t composed of mucus and airflow is critical in order to improve me dical devices and minimize hospital contaminations. Most of the experiments to assess bacteria adhesion on endotracheal tubes have been done in vivo However, the infection of the patient, his ag e, secretion rate, and many other parameters affect the bacterial adhesion and cannot be controlled in vivo Some devices have been created to mimic the trachea but they only present one type of flow (usually the airflow). Rationale Around 58,564 people died from pneumonia each year in the United States (NCHS (National Center for Health Stat istics 2004). Infections of more frequently involved organs such as urinary tract have a low rate of mortality. C ontrary to this type of common infections, VAP has a mortality ranges from 24 to 50% and can reach 76% in some cases or when lung infection is caused by high-risk pathogens. Staphylococcus aureus Pseudomonas aeruginosa, and Enterobacteriaceae are predominant organisms that lead to critical infections. Most of the patients admitted into intensive care units (ICU) develop VAP. VAP represents 47% of total infection acquired in the intens ive care units (5 to 10 VAP cases per 1000 hospital admissions and 9 to 27% of intubated patients develop VAP with an attributable mortality of 33%). To carry out experiments on endotracheal tube s with a good repeatability and a control of affecting parameters, a flow chamber needs to be designed. As the environment influences the bacteria adhesion process, it is important to comb ine the mucus flow and the air flow in order to mimic the trachea. This device provides a model representing the two complex flows that transport bacteria. This mimics an environment cl oser to the tracheal one contrary to previous

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16 experiments that only represent the airflow. Even though several experiments have been done in vivo this device allows to repeat experiments and to assess the importance of different parameters. This will lead us to several conclusions about the im portant factors that will modify and minimize bacteria adhe sion on endotracheal tubes. Objectives The first objective of the projec t was to m ake a model of a tracheal environment in the case of a diseased intubated patient. The study focused on the model of a diseased patients affected by a lung disease. As immuno-defici ent patients require a long stay intubation they are the most exposed population to any kind of infec tion. The second objective was to design and manufacture a device that would mi mic this diseased tracheal m odel to test endotracheal tube bacteria adhesion. The specific aim of the design aspect was to mimic a trachea as close as possible to the physiology. For that reason, both ai r and mucus flows had to be considered. The device also was designed to test endotracheal tube bacteria a dhesion. The bacteria adhesion process being sensitive to the ge ometry and the material surfaces, the geometry was chosen to be close to the real environment and materials ha d to have a minimum influence on it. The device had to be designed to be able to be disassembled at every experiment, every part had to be able to be cleaned and taken apart. The bacteria adhesi on experiment procedure had to be thought in order to specify the features of the device. The de sign of this device had to be thought in terms of every mechanical specification but also in its biological aspects. The final goal of this study was to set up the protocol of the experiments that would allow the assessment of the number of adherent bacteria on the tubes. The global objective of this thesis was to combine engineering with biological knowledge to mechanically mimic a diseased tracheal environment.

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17 CHAPTER 3 BACKGROUND Trachea Description The trachea is a tube with a length of 10 to 16 cm and an inner diameter from 20 to 25 mm. This windpipe allows the passage of air to the lungs. It is com posed of 15 to 20 incomplete C-shaped cartilaginous rings that allow mainta ining the airway open or collapse during the food passage. The tracheal environm ent is complex and involves two types of flow: the mucus flow and the airflow. The mucosae goblet cells are responsible for the mucu s secretion. The mucus layer is thin and covers the e ndotracheal tract. The mucus acts as a filter to exogenous particles and its flow, its chemistry, its viscosity, its thickness are important parameters for a good functioning. In a normal and healt hy case, the mucus captures bacter ia and thanks to a complex mechanism involving, hydration, ion transport, secretions and mucociliary transport, the fluid is brought to the larynx then, the pharynx and cl earance can be done e ither by coughing or swallowing. If the mucus clearan ce is affected and no longer work s properly, the bacteria or the viruses are trapped in the mucus and th e person cannot expel those (Wanner 1977). Consequently, the health status of a patient m odifies the airflow, the mucus properties and its transport so that the functioning of clearance and self-protecti on against foreign particles are affected. Mucus Description The Mucus is a viscous secreti on that covers m ost of the membranes of the endotracheal tract. It is a viscous colloid and its antiseptic enzymes (such as lysozyme) and immunoglobulins help to trap bacteria in its layer. It s pH in the throat is normally around 7.4. The mucus is a non-Newtonian fluid. It deve lops at the same time the solid and fluid rheological behaviors. This visc oelastic fluid exhibits a char acteristic relaxation phenomenon: it

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18 first stores the energy as an elastic solid and diss ipates it as the material flows as a viscous fluid. The properties of the mucus depend on many factors. This fluid is shear stress dependent. The storage modulus (G) is about 201 dyne/cm2, and the loss modulus (G) 81 dyne/cm2 at 1rad/sec whereas at 100rad/sec, G is around 275dyne/cm2 and G 1,674dyne/cm2. The mechanical properties of the mucus also depend on the health status of a person. In the case of a patient affected by Cystic Fibros is, G is about 916 dyne/cm2 and G 551dyne/cm2 at 1rad/sec and those values increase at higher rotation speed (G equals to 1,643dyne/cm2 and G to 3,148dyne/cm2 at 100rad/s) (Shah 2005). The viscoela stic behavior of tracheobronchial secretions appears to be due to glycoprotein molecules (Kieser-Nielsen 19 53). The properties of the mucus are influenced by the temperature and by this proteins concentrati on. As the elasticity is di rectly related to this constituent Autoclave and storage of mucus af fect its properties (Tippe et al. 1998). The tracheobronchial secretions are very shear sens itive which makes the establishment of a model very complicated (if the shear rates increase a reduc tion of viscosity occurs). The viscosity of the sputum of a patient affected by Cystic Fibrosis varies from 10,000 Poise at 10 dyne/cm2 to 0.1 at 1,000 dyne/cm2 (Shah et al. 2005) The thicknesses of the mucus layer have only been estimated, not quantified but the movement of the mucus varying from one species to another and it is known that its speed can be static or as fast as 35 mm/min (Tomkiewicz et al. 1995) depending on the health status of the person (gender, age, etc.). Studies have been don e to imitate its movement and suggest that the mucus does not flow evenly but preferentially co ncentrates along troughs or grooves (Agarwal et al. 1994). Sims et al. (1997) established that the thickness of th e mucus coating the trachea was not homogeneous.

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19 The mucus is generally clear and thin but its co lor can change while the body is infected as a result of trapped bacteria or reaction to a viral in fection. In the case of infected patients a dysfunctioning of the mucus clearance process is a serious problem and leads to recurrent chest infections (Afzelius and Mossberg 1980) Lung Diseases, Intubation and Effects on the Tracheal Environment and the Secretions In norm al airways, the surface epithelium is cove red by a thin mucous layer that acts as a filter and permits to trap exoge nous particles as explained above. Active antibacterial secretory proteins present in respiratory mucus usually ma intain the sterility of the airways. The mucus clearance is the collective term for epithelial water and ion transport, mucin secretion, cilia action and cough. The functioning of those parameters is disturbed when a patient is affected by a lung disease leading to several complications. Pneumonia is an inflammatory illness of the lu ngs and can be caused by an infection due to bacteria, viruses, fungi or parasi tes. Cystic Fibrosis (CF) is an inherited disease that mainly affects the lungs and causes progressive disabi lities and for some even early death (Hoiby 1982). The lungs of patients affected by CF, pneumonia, or other bronchiectasis are usually colonized by Pseudomonas aeruginosa (P. aeruginosa) Intact respiratory ep ithelium does not bind bacteria whereas injured respiratory epithelium is highly susceptible to them. Pseudomonas aeruginosa is a type species of the genus Pseudomonas It is a gram-negative bacterium with unipolar mobility. It is an opportunistic organism which frequently colonizes the respiratory tract of immuno-deficient patients (de Bentzmann et al. 1996). It is also the most frequent colonizer of medical devices such as catheters. Pseudomonas and especially P. aeruginosa can in some cases induce community acquired pneumonias, or VAP. The virulence factor of this bacterium is the Pyocyanin that contributes to the mucus transport failure (Wilson et al. 1987, 1996). P. aeruginosa is the most common bacteria that co lonize the lungs of patients affected by

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20 pneumonia. It is known that VAP caused by these bacteria has been associated with the higher fatality rates than those caused by othe r bacteria (Crouch Brewer et al. 1996). P. aeruginosa not only affect patient with CF or Pneumonia but also sever immunocompromised patients. The colonization of the airways by these bacteria is highly favored when th e patient host response decreases or when his epithelial is injured (d e Bentzmann et al. 1996). In the United-States, one in 3900 children is born with CF [1]. A patient affected by pneumonia also has difficulties to breath, and for elderly people it can lead to death. When artificial ventilation is required the patient is intubated and airflow with a certain pressure and humidity is created to assist or replace the spontaneous breathing. In the case of chronic diseases, the patient may have to be intubated for a l ong period of time. When it is the case, several difficulties occur such as VAP (it can occur only 48h after the intubation). The endotracheal tube opens a free passage to bacteria into the lower parts of the lu ngs. Cook et al. (1998) studied 1,200 patients and assess the time-dependent risk factors for ventilator-a ssociated pneumonia and determine the impact of a long intubation on ICU patients. Onehundred and eighty-six of those patients were first excluded for different r easons: 85 were discharged, 71 died, and 30 had pneumonia in the first 48 hours. The results s howed that 17.5% of the 1014 patients left who were free of pneumonia at the beginning develo ped ventilator-associated pneumonia after 9.0 5.9 days.

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21 Figure 3-1. Prevalence of VAP related to the number of days on mechanical ventilation (De Queiroz Guimares and Rocco 2006) When a patient is intubated, he is usually sedated or even paralyzed and cannot cough. This leads to aspiration of inf ected oropharyngeal contents thr ough the open vocal cords. The secretions pool accumulates above the inflated endotracheal tube cuff and as the patient is mechanically ventilated the pressure of the cuff ch anges. It deforms and allows secretions to be transported around the cuff by capillary action (Rum bak, 2002). Also, when the cuff is deflated after several weeks, to allo w correct positioning for tracheotomy the pooled secretions and bacteria can disseminate through the lungs. The shear forces of the gas flow which can be as high as 200mL/s depending on the ventilation mode also affect the accretions on the luminal surface (Koerner 1996). Tracheal intubation is then reco gnized as a major risk factor for pneumonia (Adair et al. 1999). Drugs are administered to ICU patients. Th ey prevent the mucus from its normal motion and lead to a defici ency of mucus clearance. The only parameters that act on the mucus layer are the gravity and the airflow that comes up from the tip of the cuff tube. The mucus flows at 1.4 2.4 mm/min compared to 1 0.2 4.4 mm/min for healthy persons (average calculated for 50 healthy patients and 45 patients affected by a chronic obstructive pulmonary disease, Morgan et al. 2004). Patients affected by VAP (%) Number of days under mechanical ventilation

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22 The mucus viscosity and elasticity increase when a patient is affected by a lung disease. It induces a problem of cleaning of the airways. Un der pathological conditions, the mucus is over secreted and inflammation occurs; this will favor the colonization by Pseudomonas aeruginosa. CF leads to an abnormally production of thick, st icky mucus, due to the faulty transport of sodium and chloride (salt) with in cells lining the l ungs. The submucosal glands contain serous tubules and acini that secrete salt, water, and various antimi crobial proteins. The serous secretions pass through mucous tubules, where viscous glycoproteins are added, and then into a collecting duct and onto the airway surface. Active salt and water secret ion by serous epithelial cells is believed to involve Cltransport by the cystic fibr osis transmembrane conductance regulator protein driving water transport. Subm ucosal gland secretions are important for the generation of a thin mucus layer and for the crea tion of an environment that inhibits bacterial colonization. Abnormalities in submucosal gland secr etions have been proposed to contribute to the airway pathophysiology in CF. CF transmem brane conductance is expressed in serous epithelial cells of submucosal gla nds more strongly than in othe r tissues of the airways and lung. It was postulated by Jayaraman et al. (2001) that the salt content and viscosity of submucosal glandular secretions in CF are abnormal and lead patients to several complications. Over secretion of mucus in the airways leads to c ongestion and an increase d susceptibility to lung infections which are the major cause of morbidity and mortalit y among people with CF. Chronic bacterial infections of airways can also be caused by mucus adhesion, formation of mucus plaques and ultimately mucus plugs. When the airw ay surface becomes severely dehydrated, the viscous, adhesive mucus layer can interact with cell surface mucin resulting in adhesion of the mucus and decrease of the clearance, and ulti mately the formation of plaques and plugs of concentrated mucus (Scott et al. 2006).

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23 Bacteria and Adhesion Pseudomonas aeruginosa is gram -negative, aerobic, rod-shaped bacterium (Ryan and Ray 2004). Its high mobility is due to its rod-shape and its flagella that it uses to swim. The speed of the bacterium between tumbling can vary from 1 to 10m/s depending on the media (viscosity, temperature, pH...). In the case of the mucus, as th e shear rate modifies its viscosity, the flow rate is an important parameter in terms of how it can modify its mechanical properties. The mobility of a bacterium depends on the medi a properties and is then influe nced by the flow rate of the mucus or any kind of media. Engelmann (1881) showed that the bacteria do not usually move randomly thanks to chemotaxis. Bacteria are able to change their movement in response to the gradient of certain chemicals and also by communicating with others. Bacteria are cooperative and self-organized. One type of bacterial cell-cell communication is referred to as quorum sensing (Golding et al. 1998), and occurs only if bacteria are at high density. Bacteria move in a certain direction following different parameters they are able to feel. They move for instance toward a region where there are more nutritive substances. Bacteria grow in biofilms that offer adhe ring protection against antibiotics and other environment attacks (Duguid et al. 1992). The bacterial adhesion is a complex mechanism that can be described by a two-phase process: one is initial, instantaneous a nd physically reversible, the other is a time-dependent and irreversible mo lecular and cellular phase (Marshall et al. 1985). The first phase can be called the physicochemical interactions between bacteria and material surface. Bacteria move towards the surfaces thanks to different forces: long-range interactions (non-specific, distances < 150 nm) which are fu nctions of the distance and free energy, and short-range interactions (< 3 nm effective when the bacteria and the surface come into contact). Those two types of interactions allow the first st ep of adhesion, an initia l attachment between the

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24 surface and the bacteria. The second phase can be called: the molecular and cellular interactions between bacteria and material surfaces (An. and Fridman 1998) The environment characteristics such as the temperature, the time of exposure, the bacterial concentration, the flow conditions, the chemical treatment or the presence of an tibiotics affect bacter ia adhesion. The flow conditions also influence the number of attached bacteria (Duddridge et al. 1982, Dickinson and Cooper 1995) but also the biofilm structure and performances (Stoodley et al. 1999, Klapper et al. 2002). Surface Chemical Compositions Influencing Adhesion Different param eters related to the surface composition influence bacterial adhesion and proliferation. The surface r oughness affects the adhesion. Indee d, irregularities of polymeric surfaces promote bacterial adhesion and biofilm deposition (Katsikogianni and Missirlis 2004). Bacteria adhere and colonize on porous su rfaces and grooved and braided material preferentially. The surface confi guration is also an important parameter in the adhesion mechanism. Finally, the surface hydrophobicity or wettability seems to affe ct the process of adhesion. The hydrophobicity of a material surface is mainly measured by contact angle measurement. It was shown that hydrophilic materials are more resistant to bacterial adhesion than hydrophobic material. The chemical properties of both surfaces: bact eria and material have an impact on the adhesion process (Van Loosdrecht et al. 1989). The surface charge influences the adhesion (Taboada-Serrano et al. 2005). The surface characte ristics are one of the main parameters to control and to diminish the bacteria growt h, hence the research of a topography preventing bacteria adhesion (Koerner 1996).

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25 Bacterial Properties Influencing Adhesion The bacterial hydrophobicity can be obtained by contact angle m easure ment. Bacteria with hydrophobic properties generally prefer to ad here to hydrophobic material surfaces. The bacterial surface charge is also an importa nt data to assess to understand the adhesion process of each type of bacter ia. Most particles acquire a surf ace electric charge in aqueous suspension due to the ionization of their surface groups. The surface charge of bacteria varies according to their species and is influenced by th e growth medium, the pH and the ionic strength of the suspending buffer, bacterial age, and b acterial surface structur e. The surface charge attracts ions of opposite charge in the medium and results in the formation of an electric double layer. The surface charge is usua lly characterized by the isoelect ric point, the electrokinetic potential (or potential), or electrophoretic mobility (Cerca et al. 2004, VadilloRodriguez and Logan 2006) Biofilms The for mation of biofilms can contaminate ever y kind of surfaces from the food to patients under medical treatments or catheters and the consequences can be severe. Biofilms are a community of bacteria embedded in a polymeric matrix that adheres to the surface (Carpentier and Cerf 1993, Costerton et al. 1987). Extracellular poly meric matrix contribu tes to the biofilms stability and adhesion (Bellon-Fontaine and Briander 2000). Micro-organisms usually build biofilms and most of the materials in contact with a natural fluid are rapidly covered by bacteria. Despite the fact that th e first scientific studies carried out on biofilms were done in 1943, this is only in the 70s that it was realized that biofilms were abundant in natural medium (Costerton et al. 1987). Bacteria in biofilms can get different chemical and physical properties from their homologs present in aqueous solutions. It has been proved that bacteria growing in biofilms

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26 acquire more resistance to different stresses such as dehydration, lack of food, or antibiotics than isolated bacteria in solu tion (Morton et al.1998). Bacteria biofilms start their formation once some individual bacteria attached to the surface. The environmental factors as describe d in the previous paragraph (pH, nutrient, viscosity of the environment) but also genetic factors (the presence of genes encoding motility functions, environment sensors, adhesins) are rele vant in terms of ability to attach or not to a surface. The bacteria properties such as hydropho bicity and compatibility with the surface are important factors in this first adhesion step. Once the first adhesion stage is reached the bacteria grow in a monolayer and form microcolonies. Du ring the biofilm growth, bacteria properties evolve and undergo several change s that later allow them to be more resistant to any kind of environmental attacks. One of these change s is the exopolysaccharide matrix production. Biofilm Formation The biofilm formation follows several diffe rent steps before reaching its mature architecture. The first step is the motion and tr ansport of micro-organisms. The bacteria can swim in the medium thanks to their flagella. Th e medium properties then affect its ability to move. Thus, non-swimming bacteria have a redu ced capability of growing in biofilm. One percent of the genome is devoted to the flagellar function for most of the gram-negative bacteria. The second step is the initial a dhesion to the surface. Once the first bacteria adhere to the surface, the synthesis of extracel lular substances such as exopolysaccharide matures the biofilm. Allison and al. (1987) showed that a bacteria or a mutant that are unable to synthesize exopolymeric substances cannot adhere to any support whereas one single bacteria producing exopolymers is capable of forming microcolonies The last stage is the colonization of the surface by multiplication of the adherent bacteria Thereby, the biofilm can keep growing and spread into uninfected areas and even some of th e bacteria can detach to colonize other regions

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27 (Figure 3-2. Kraigsley and al.). Thus, the colonization of surfaces particularly depends on the motion of bacteria toward the support. This transpor t is not only based on th e bacteria self ability of motion but also on physical and microbiologic factors such as the Brownian motion, gravity (Bryers 1987), hydrodynamic characteris tics of the fluid (static, lami nar or Turbulent) (Korber et al. 1995). Studies have shown that mutants of P. aeruginosa non-mobile are unable to form biofilm on PVC contrary to mobile P. aeruginosa Figure 3-2. Biofilm formation and growth (OToole et al., 2000) Forces Governing the First Step of Adhesion Once bacteria are close to the surface they can adhere by a process of physical and chem ical interactions (Electrostatic interactions electrodynamics interacti ons or Lifshitz-Van der Waals, interactions donor/ receptor of electrons or Lewis law on base/acid, interactions due to the Brownian motion of particles). Interaction and Complementar ities betw een Bacteria As it was mentioned before, bacteria communi cate with each other and cooperate. When a micro-organism develops, grows it changes th e environment properties. Indeed, a microorganism modifies its environment by consum ing nutrients, producing wastes that can be appropriated for the growth and development of ot her type of micro-orga nisms. Siebel et al. (1991) showed that a single species biofilm thickness was much smaller than the thickness of a biofilm composed of several species. (K. Pneumonia and P. aeruginosa isolated show a biofilm

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28 thickness of 15 and 30 micrometers respectively whereas the thickness of a biofilm containing both species could reach 40 micrometers). Pseudomonas Biofilms and Anti biotics Resista nce As it was explained above, the growth of biofilm is favored by the production of extracellular polymers. A significant number of Pseudomonas produce exopolysaccharides and then are able to build a mature biofilm. The exopolysaccharide prevent Pseudomonas from being phagocytosed by mammalian white blood cells. Pseudomonas can adapt to different types of environments as they are able to metabolize vari ous kind of nutrients. Then they can thrive in every unexpected place even in very harsh conditions (Lambert and Drenkard 2002). P.aeruginosa has a great resistance to antibiotic. Fi rst, the growth in biofilm makes them harder to eradicate thanks to penicillin or majo r antibiotics. To struggle against antibiotics attack they use the efflux pumps (Stewa rt and Costerton 2001) called ABC transporters which expel the antibiotics before they can st art acting. Not only intrinsically resistant they also undergo mutations of their genes to improve their resistance. Adhesion Tests Dif ferent techniques exist to characterize the bacterial adhesion and can be separated in two groups: the static and the dynamic methods. Static Adherence Method Three m ethods to assess the number of adhere nt bacteria under static conditions are described here; several adaptations of those me thods exist. A piece of substratum such as a catheter is immersed in a microbial suspension and a batch adhesion process is allowed to occur. After exposure, the substratum is washed for re moval of non-adherent bacteria and then adhering bacteria can be enumerated. Ther e are different methods to count the bacteria, the most common might be the image analysis method using a phase contrast microscope co upled to a 3CCD video

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29 camera that acquires images with a specific re solution and that uses automated enumeration software to count the cells (Cer ca et al. 2004). The adhered bact eria can also be enumerated using a fluorescence microscope by counting 10 differe nt fields colored by acridine orange (the random surface of the field chosen for the obs ervation has to be determined) (VadilloRodriaguez and Logan 2006) Another method consists of using squares of th e studied material, places them in plates containing cell suspension at an optimal concentrat ion in saline solution. In itial adhesion to each substratum is allowed to occur during an optimal adhesion time at 37C, in a shaker at a certain shaking speed (rpm) (Cerca et al. 2004) The last method described is the method that assesses the rate of adhesion by measuring the decrease of OD660 (Optical density at 660 nm wavelength). Prior to the adhesion experiment, the linear relationship between cell number and optical density at 660 nm wavelength is confirmed under all experimental conditions. Then, a certain amount of cell suspension prepared as described by Terada et al. (2006), was added to a beaker. The concentration of the cell suspension is set. Sheets of the material studied are cut into 0.025 cm. Each sheet is immersed in a b eaker containing a cell suspension. The assessment of the rate of adhesion is measured by the decrease of OD660 of each cell suspension. The observation of bacterial adhe sion is done by scanning electron microscopy (SEM). The relevant parameters of those static methods are: the adhesion time (time of exposure), the velocity (rpm) of the shaker the container size, the solution volume, the counting method and the washing method (immersion back and forward a certain amount of times, or by dipping the samples, rinsing solution (dematerialized water, PBS...). The re sults obtained by static method

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30 are the amount of cells adherent. However, the washing method, the velocity of the shaker and the adhesion time are three parameters very influe nt on the results of the adhesion tests and that is the reason why a dynamic method under flow conditions seems to be more relevant. Dynamic Method Parallel-plate flow chamber and im age anal ysis: images taken from the center of the bottom of the plates with a 3CCD video camera mounted on a phase-constant inverted microscope equipped 40x ultra long working distance objective. All tubes and the flow chambe r are filled with a saline solution. Th e saline solution circulates until the stationary conditions are obt ained (the choice of the regime determines Reynolds Number). The flow is established by th e hydrostatic pressure a nd a peristaltic roller pump is used to make circulate the suspension. During the time of circul ation of the bacterial suspension images are captured. After the enumer ation of adhering bacteria an air-liquid interface passes through the device so that the amount of cells re moved is determined (Roosjen et al. 2003). The parameters of the dynamic methods are: th e time of circulation, th e temperature of the room or of the flow chamber, the time interv al between two captured images the Reynolds number (regime of the flow). This type of expe riment provides the number of adherent bacteria at a stationary end-point and the initial deposition rate can be assessed during the first minutes of adhesion test. Comparison of the Static and Dynamic Methods The m ajor inconvenient of this static analysis is the lack of accurac y. Indeed, every static method provides a qualitative approach. Only the amount of cells adherent at the end of the essay can be assessed and the experiment depends on too many parameters. The adhesion time (time of exposure), the velocity of the shaker if a shaker is used and the way the sample is washed are the

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31 most important parameters that contribute to the technicality of comp aring different static adhesion tests. For those reasons, an experiment using flow conditi ons (closer to the reality in most of the cases) can provide a quantitative approach of bact erial adhesion. The experiment under flow conditions consist usually of using a pa rallel-plate flow chambe r. This is the most common configuration as it is simple to make and several flow conditions can be set up. Tracheal Models Different mechanical tracheal m odels have b een developed to test the properties of the endotracheal tubes. Most of them are simple a nd are usually composed of a clear rigid, plastic tube that mimics the trachea connected to a mechanical lungs Figure 3-3. Horizontal trachea l model (Weiss et al. 2007) The studies using a tracheal model and an endot racheal tube can have different purposes from determining if inspiratory pressure from intermittent positive pressure ventilation may be sufficient to inflate the cuff (Guyton et al. 1991), or showing that ther e is a leak of fluid past the tracheal tube cuff (Dullenkopf et al. 2003, Young et al. 1997). To test the bacterial adhesion the experiments carried out are usually on animal or comparisons between in tubated patients in the hospital. (Inglis et al.1989, 1998, Adai r et al. 1999, George et al.1998)

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32 During the intubation, the cuff does not prevent infection and bacterial colonization of the lower trachea. A certain amount of patients in hospital are examined to get data on biofilms growth adhering endotracheal tube and how different parameters influence the adhesion. For that purpose, several experiments are carried out on E TT collected from intensive care patients that are under different type of ventilation, humidity system and heat and moisture exchangermicrobiological filters. The results usually show that quantitative culture of biofilm showed viable counts of up to 106 CFU/cm of tube length and P. Aeruginosa has the highest viable count. Most of the studies reveal that the growing biofilms accu mulate on the inner surfaces of the ETT and some contaminated particles attached to this layer can be spread out during the mechanical ventilation. The inside part of the tu be also seems to be contaminated (about 50% of the surface can be contaminated depending on how long the patient is intubated). Consequently, many parameters affect the biofilm growth: time of intubation, the amount and the viscosity of secretions (the more the patient s secrete tracheal mucus the more he gets infected apparently), the humidification, the pH... The qualitative approach consists of removing the ETT from the ICU patients. Each tube is sampled with cotton sw abs (different parts of the tube (tip and cuff) and specimens are plated on 5% horse blood ag ar. After an overnight incubation standard bacteria identification is done. Th e quantitative method consists of collecting biofilm at the level of the cuff. Biofilm are homogenized in 2% N-acetylcysteine (in 0.15 M saline, pH 7). The samples are diluted and spread on agar plates. Th e viable colonies can be counted by automated colony counter. The amount of biofilm in tracheal t ubes can also be assesse d. Inglis et al. (1989) have found that 30 out of 40 tubes examined have a dry weight of biof ilm greater than 50mg. Those studies also show that P. Aeruginosa account for the highest viable counted colony isolated from the tube. Bacter ia adhesion on ETT was assessed directly on patients under the

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33 same type of ventilation. The mucus propertie s changing different factors will influence the adhesion process. Models of trach ea have been created using a horizontal model with airflow. The mucus is not mimic, only a fluid is used to characterize the leak around the cuff. Consequently, a model combining both flows seems to be relevant.

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34 CHAPTER 4 DESIGN OF A TRACHEAL MODEL The design of this tracheal flow m odel has to be thought in terms of physiology, mechanics, feasibility and the practical aspect of the device. It has to complete several specifications: the parameters relative to the tracheal environment (geometry of a trachea, creation of two flows (mucus and air) of a diseas ed patient, model of l ungs), to the bacteria adhesion test (material compatibility, special assembly), mechanical design (the way of manufacturing, designing the parts and assembling them). Specifications The geom etry of the device has to be as close as possible to the reality. The main shape has to be cylindrical in order to represent the windpipe of the trachea. The mucus flow has to be transported by gravity action. The created mucus fl ow has to be in a thin layer (around 200 to 300 microns) covering the inner part of the trach ea. When a patient is intubated and needs intensive respiratory cares, he is not laid on hi s bed but inclined in order to help the mucus clearance (gravity effect). To reproduce this flow, the easiest way is to let the gravity act naturally. The mucus flow rate is around 1mm/ min but depending on the persons health, the type of infection this rate can change a lot. In the case of intubated patients, as the mucus is getting thicker and thicker with the infection, it is important to use a natural pool of subglottic secretions in order to get the right visco-elastic properties and reach similar flow conditions as in vivo The mucus has to flow naturally in a pipe and cannot be sucked out of the device (the flow naturally goes in the lungs and is not sucked out ). Once it has flowed on the wall, it has to be stored in a reservoir to prevent too mu ch spread of bacteria in the air. The airflow created has to be as close as po ssible to the one used in intensive care units. The device used has to simulate a spontane ously breathing patient. As the cuff of the

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35 endotracheal tube is not a perfect seal with the trachea, air have to come up and be able to be released in the atmosphere as it is in reality. At the bottom of the device, the lungs have to be modeled. The created device must have no leak in the mucus reservoir to prevent the spread of infected mucus. Not having mucus leakage is an im portant factor but also the bottom part of the device has to be airtight in order not to let the air co ming out (lungs are air tight). The temperature has to be maintained at 37C and the materials chosen has to be autoclavable and non-cytotoxic. Design of the Tracheal Model and Method General Principle of Functioning The trach eal model of a diseased patient that we chose is composed of a mucus layer covering the wall of a lexan t ube, the endotracheal tube insi de the trachea and the airflow plugged to the ETT to mimic the conditions of the intensive care units. Figure 4-1. Schematic of the diseased patient tracheal model Gravit y Mucus layer T rachea Endotrachea l tube Airflow

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36 The trachea is mimicked by a lexa n tube of inner diameter of 1 inch (25.40mm) and outer diameter of 1.25 inch. Those dimensions are chos en from a mechanical and a physiological point of view. The advantages of such a tube are that the dimensions ar e standard and the material is biocompatible. It is a little higher in diameter than the mean diameter of a trachea (25.40mm compared to a real diameter of a trachea from 22mm to 25mm) but it was chosen in term of feasibility and standards. In this device metric and British units are mixed in order to get standard parts. The airflow was created by an appropriate ai r flow device found in the industry. The air went through the endotracheal tube and filled a balloon (Breathing bag, 0.5 Liter) that mimics the lungs. As the cuff does not create a perfect seal with the lexan tube, to allow the air to escape, a hole is added in the top part of the lexan tube. This hole acts as the leak that naturally exits in the throat when a patient is intubated (not the whole air goes down the lungs ). This hole is also used to pull out the tube fr om the inflated cuff balloon. The mucus flow is generated by a Harvard Appa ratus Syringe Pump m odel It fills a mucus tank that has a cylindrical shape with a conical shape inside. Once the cone is full of mucus it overflows in the 300 micrometers groove (gap between the lexan tube and the mucus tank). The syringe pushes at the beginning to get over the surface tension that exists between the fluid and the wall and then the gravity acts on the thin layer. The mucus flows on the lexan tube wall. Thanks to the cone-shape of the top mu cus tank, the flow is more homogeneous. At the bottom of the lexan tube the mucus flows th rough two oblong grooves and enters the main mucus reservoir. The main mucus reservoir di mensions are designed considering the worst conditions. The mucus is assumed to be a Newtonian fluid, and the flow ha s a linear distribution (linear model) with a no-slip conditions at the walls.

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37 Calculations of the Dimensions The m ucus is considered as a Newtonian flui d for the assessment of the flow conditions. The mucus viscosity is considered to be 6.5Pa.sec and the density 1000kg/m3. The geometry of the trachea model is showed Figure 4-2. The radius of the mucus reservoir is chosen equal to 37.5 mm. The mucus starts to fill the cylindrical bottom part and then flows inside the mucus reservoir through the two grooves. The maximum hei ght of the mucus is 10mm in order not to fill the balloon. For a radius of 37.5mm of the mucu s reservoir, the capacity is equal to 32.6mL (Figure 4-3). The mucus tank on the top has a volume of 4.9 mL, knowing that the shape is conical. As the gap between the lexan tube and the mucus co nical tank is 300 micromet ers it is assumed that the mucus layer flowing has the same thickness (in reality this is not the case). The area of mucus called A1 is equal to 1.189x 10-5 m2. Considering the following fluid mechanics model of linear profile the wall shear stress and the maximu m flow rate of mucus ar e assessed under those particular assumptions. Figure 4-2. Tracheal model Mucus Thickness: 300m ID Tube = 25.40 mm A1 Mucus Area

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38 Figure 4-3. Mucus reservoir geometry (unit: millimeter) The wall area of a thin layer of mucus in m2 is equal to 0.1596 x dz where dz is the height of this layer (Figure 4-4). It wa s considered that z equals one me ter to simplify the calculus. The mucus layer weight is calculated assuming that the density of the fluid is 1000 kg/m3 and is equal to 0.1167 N. Figure 4-4. Wall shear stress .1. 2..tube A g R z (4-1) Where A1 is the mucus area, Rtube is equal to 0.5 inches, g is the acceleration of the gravity, and Rho is the mucus density. Assuming a linear and fully developed profile of the mucus flow (Figure 4-5), the veloc ity has the following expression: Wall Area of a mucus layer A2 g z Lexan tube, the mucus flow on the wall Mucus reservoir The mucus flows through the grooves and enterthereservoir Maximum height of the mucus

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39 uayb (4-2) The boundary conditions give the expression of the velocity u (no-slip condition at the wall). Knowing the shear stress, the maximum velocity can be assessed. u y (4-3) The maximum velocity was equal to 6.75x10-5 meter per second and the mean flow rate 0.40 micro liters per second. To fill the reservoir it took approximat ely one day. In order to run the experiment longer, the mucus storage could be empty out thanks to the valve under the plate. Figure 4-5. Shear stress on th e mucus layer (Wikipedia) Description of the Different Parts Base The base is designed so that the breathing bag can be connect ed under it. Four tapped holes are connected to adjustable feet McMaster -Carr 3/8-16, Part # 23015T66. Two grooves are manufactured to insert o-rings with AINSI dime nsions. This provides the no mucus leakage in the reservoir. The -28 hole is used to connect a manifold. When the experiment is finished the reservoir can be empty out easily by opening this valve or simply remove the screw. Lexantubewall

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40 Cylindrical Bottom The mucus flows in the cylindrical bottom first and thanks to the tw o grooves enters the bigger reservoir (Figure 4-6). Figure 4-6. Cylindrical bottom Mucus Tank The mucus tank is designed to play the role of a small reservoir. Once it is filled up (the angle of 45 makes the shape of the reservoir) it overflows and as the gap between the lexan tube and the conical tank is approximately 300 m, the mucus is pushed between the walls. The complete drawings of the device that were used to manufacture this tracheal flow chamber are available appendix C (the assembly method is described appendix E) and an exploded view (Figure 4-8) shows the device. 2 Grooves at 180 to leave the mucus flow naturally in a bigger reservoir (the mucus storage)

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41 Figure 4-7. Mucus tank and top cover

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42 Figure 4-8. Exploded view of the device

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43Mucus and Air Tightness of the Device The top of the device should not have any mucu s leakage. The top mucus tank is filled and the syringe pump has to apply a higher pressure to overcome the wall friction and surface tension between the mucus and the small gap between the lexan tube. In order no t to have leaks it was important to put o-rings between every part. Figure 4-9. O-rings position 3 O-rings Drilled screws O-rings

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44 The mucus storage also had to be without leak of air and mucus in order to keep a constant pressure in the breathing bag connected to the bottom of the device. Modifications In order to make the experiments repeatable the lexan tube has to be replaced each time. The design is modified to have lexan tubes dispos able and press fit in the device. Two parts are designed to fulfill this requirement. Also, future modifications would be the design of different sizes of drilled screws to test other endotracheal tubes size. The part Diam 14 airflow inlet can be easily manufactured with different diameters. This part would adapt the de vice to other type of endotracheal tubes. Figure 4-10. Adjustable endotracheal fitting

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45

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46

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47 CHAPTER 5 CHARACTERIZATION OF THE FLOW The device has to be ab le to cr eate a flow of mucus that covers the wall of the lexan tube. The concept of the device is to mimic a trachea w ith a mucus layer that covers the inside of the tube as it covers all the tracheal tract membranes in reality. The objective of this chapter is to validate the concept of the tank with a conical sh ape that overflows to co ver the wall of a mucus layer. Materials To mimic the mucus, dextran produced by Leuc onostoc mesenteroides (Strain No. B-512, Industrial Grade, Average Molecular Weight 5,000,000 to 40,000,000 Daltons from Sigma Co.) has been diluted in water. Dextran is a complex, branched polysaccharide made of many glucose molecules joined into ch ains of varying lengths. The solution used is com posed of 25g of dextran for 100ml of water. The viscosity is measured thanks to a Brookfield Viscometer (Fig. 5-1), with a cone at 3 CP52 and a sample of 0.5ml. 30ml syringe of this solution provides the flow to the device using a Syringe Pump Harvard Apparatus model 33. Figure 5-1. Brookfield viscometer

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48 Methods Once the dextran solution is characterized (range of viscosity), the solution is separated in two beakers. One beaker is used to coat the lexan tube the othe r to characterize the flow. Food coloring are added in each beaker to make the difference between the flow coming from the top tank and the coating. The flow rate is first set at 1ml/min so that the so lution can rapidly fill the tubes. Once the surface tension betw een the top tank and the lexan tube is overcome the flow rate can be diminished and set following the diffe rent results given in the appendix-d. Results Dextran Solution The dextran solution used for the primarily results is a 25% concentration. The behavior of this solution seems to be close to the mucus one : elasticity and viscosity. Dextran solutions at high concentration do not present a Newtonian behavior. Figure 5-2. Dextran solution

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49 The dynamic viscosity of the solution was a ssessed using the Brookfield viscometer. As the solution does not present a Newtonian behavior the viscosity is shea r dependent. A mean of different experiments has been done. It was shown that over 10 rpm strings appear and the viscometer cannot stabilize. Figure 5-3 show s the mean behavior of this solution. 0 1000 2000 3000 4000 5000 6000 7000 8000 1251 0 0 512 55 Shear Stress (sec-1) Angular Velocity (rpm)Dynamic Viscosity (cP) Figure 5-3. Mean dynamic viscos ity of 25% dextran solution Following the study of A.Tippe et al. (1998) done on the viscoelastic properties of canine tracheal mucus, the mucus range is from 1600cP to 84000cP (for an angular velocity going from 1rad/sec to 10rad/sec). The dextran solution seem s to mimic well the mucus properties and this is the reason why it was used for those experiments. Homogeneity and Characterization of the Flow The flow rate was first set at 1ml/min, the lexan tube was coated with the dextran solution and green food coloring was added to it. The red flow characterizes the flow coming from the top

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50 tank (the one that will later cont ain bacteria and that will mimic a real intubated patient trachea) (Figure 5-4). Figure 5-4. Experiment showi ng the beginning of the flow

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51 Figure 5-5. Flow around the cuff tube The flow is covering the wall of the lexan tube as it was expected and depending on the cuff pressure the flow leak around. This fact will be interested later to characterize the rate of adhesion around the cuff (the most sensitive area of the ETT to bacteria adhesion). Figure 5-6. Mucus reservoir flow

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52 Once the lexan tube is covered by the layer of dextran solution it flows in the mucus reservoir through the two grooves that were designed in the cylindrical bottom part. The flow rate set at 1ml/min or 500ml/min is too high to be relevant in term of physiology but it makes the flow visible. Experime nts carried out with lower flow rate, close to the real one (20L/min) provide a thin layer of mucus on the wall as show fig. 5-6. Figure 5-7. Aspect of the coat ing for different flow rates

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53 CHAPTER 6 BACTERIA ADHESION TEST Materials The experiments were carried out using a syringe pump Kd Scientific (KDS 100 SingleSyringe Infusion Pump) that provided the mucus flow. A 30mL syringe was connected to a stop cock. The stop cock manifold is composed of one input and three output connections plugged to 3 tubes (OD 1/16) that can be inserted in the drilled screw holes on the top cover. A KimberlyClark Microcuff Endotracheal t ube (ID 8.0mm, OD 10.7mm) was used for this preliminary experiment. The endotracheal tube was connected to the hose of an Air Cadet Vacuum Pressure Station (Cole-Parmer) to mimic the air flow. In order to control the pressure of the cuff, the valve at the end of the cuff tube should be connected to a Pressure easy Cuff (PressureEasy, Cuff Pressure Monitor, Medex Inc.). However, this device was not available for this experiment, therefore the syringe connected to the valve was us ed to keep a constant pressure. The bottom of the device was connected to a breathing bag 0.5 Liter fitting 22mm (Breathing bags A-M Systems, Inc). A more in depth explanation is gi ven in Appendix E which explains the details of the experimental protocol.

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54 Figure 6-1. Experimental set-up Syringe Pump Kd Scientific Device + Endotracheal tube Breathing Bag Air Cadet Vacuum Pressure Station Syringe to control de pressure of the cuff balloon

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55 Methods Egg white was used as a mucus simulant and cu lture media. In future studies, a pool of subglottic secretions would be co llected directly from patients a nd sterilized by gamma radiation. To overcome surface tensions at the beginning of the experiment, the lexan tube and its connector are coated with egg whites. Half of the egg whites collected were used for the coating (100mL) and the other half for the culture media. The device was sterilized by autoclave and by dipping parts in 70% ethanol and later air dried. The device was assembled in sterile c onditions under a biologi cal hood as explained Appendix E. Twenty four hours prior to the experiment, a crystal of Pseudomonas aeruginosa was placed in 5mL triptic soy broth (TSB) media in a 18x150 mm sterile culture tube with screw cap. The culture tube was placed in a waterb ath/shaker at 37C and 150 rpm for overnight growth. The bacteria were counted using optical density spectrophotometry method following the growth curve of Pseudomonas aeruginosa The cultivation process t ook place in the incubator at body temperature of 37C. The air flow rate was 14.1L/min provided by the Air-Cadet ColeParmer. The air flow rate was imposed by the device we used; however, it is higher than the respiratory minute volume (5 to 8 L/min for a normal person (Wikipedia)).The mucus flow rate was set at 20L/min. The optical density of the solution of TSB and Pseudomonas aeruginosa used was 0.175. Five milliliters of this media we re mixed with 30mL of egg white in a sterile conical tube, and then 30mL of this solution were introduced in a sterile syringe for the experiment. Bacteria Count After 24 hours of incubation, the tracheal fl ow chamber was removed from the incubator and placed under the hood. Wearing sterile gloves, the top cover was slowly disconnected from

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56 the lexan tube avoiding contact with the endotracheal tube. Six 1 cm-rings of the endotracheal tube were cut (two at the cuff level, two above and another at the tip) and placed in sterile conical tubes containing 15mL of PBS. The biofilm was homo genized by vortex mixing 1-cm rings of tubing in 10mL of biof ilm surfactant solution (Medtroni c Xomed Inc.). Viable counts were made by serial dilution-agar plate techni que after overnight inc ubation in air at 37C. Results and Discussion The experiment was carried out for 24 hours. The biofilm of P. aeruginosa grew in the device with increased expression around the cuff balloon as it is in vivo The tracheal model therefore resembles physiological conditions. Th e air coming upward from the balloon and leaking around the cuff produced a shear stress around the cuff; this seemed to increase the adhesion. The airflow dried the egg white and in th e case of intubated patient, the air flow in ICU would also dry the tracheal tract and leads to severe dehydration. Hence, the model seems to be physiologically relevant. Figure 6-2. Dried egg white in the tracheal flow chamber

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57 As a last step, 100L of the contaminated egg white contained in the syringe were plated and showed the growth of the bacteria and the green aspect of the biofilm on the agar plates (Figure 6-3). Dilutions of the solution contained in the syringe allowed counting the number of bacteria that were inserted in the device. It showed that P. aeruginosa grew in the egg white media. Figure 6-3. Agar plates with P. aeruginosa isolated from the egg white contained in the syringe The ETT was cut in six 1cm-rings, the first ETT segment was cut at the tip, segments 2 and 3 were cut at the cuff level, segments 4, 5 and 6 were cut above the cuff. The aspect of the biofilm surfactant solution that fixed the biof ilm changed depending on the segment observed. The tip seemed to have less biofilm than the other segments. The solution containing segment 3 of the cuff looked more flocculant (Figure 64). The adherence of th e egg white was more important around the cuff (flocculant aspect due to the proteins). The egg white flowed from the top of the device and coved the wall of the lexa n tube. The cuff balloon was in contact with the lexan tube and more susceptible to be contaminat ed. On the other hand, the tip was not directly in contact with the egg white as the cuff blocked the flow off (few leaks). Dilution 10-1 Dilution 10-2 Dilution 10-3 Dilution 10-4 Dilution 10-5

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58 Figure 6-4. Aspect of the biofilm su rfactant solution containing ETT segments The dilutions of those solutions containing the ETT segments on agar plates did not yield any results. The bacteria did not grow as much as expected. B acteria count was only possible on the agar plates that were inoculated without any dilution of the biosurfactant solution. Figure 6-5. Agar plate showing the bacteria growth for each ETT segment 1 2 3 4 5 6 28 CFU/cm Above 4000 CFU/cm 600 CFU/cm 90 CFU/cm 850 CFU/cm 725 CFU/cm

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59 The number of adherent bact eria was maximum on the segment 2. This segment was located around the cuff balloon in contact with the lexan tube. Th is result was expected however, the segment 3 should have also showed a signifi cant bacteria growth. The segment 3 presented a lower growth that could be attrib uted to the cutting me thod. It is crucial to not alter the biofilm and the manipulation of the ETT needs more practice to be accurate. Overall, the bacteria growth was not as prom inent as expected. This can be attributed to the temperature and humidity of the airflow. Th e Air Cadet vacuum pressure station was placed outside the incubator and the air blowing in the device was cold and could not be successfully humidified. The egg white might have dried at an increased rate and did not properly transport the bacteria on the wall of the lexan tube. Th e air flow should be produced by a breathing simulation module and the tube should go throu gh a water bath to humidify the air before it enters the device. Despite of these issues, due to a lack of time, it is clear that the device performs as intended. The device along with the developed experimental pr otocol provides a means for assessing endotracheal bacter ia adhesion under physiological conditions. The preliminary adhesion data are promising but require additional work.

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60 CHAPTER 7 FUTURE WORK AND CONCLUSION Type of Experiments Real mucus will be used for future experiments. If it is sterilized by gamma radiation, only one kind of bacteria can be injected in it (i.e. P. Aeruginosa ). Experiments may also be run with the natural subglottic po ol of secretions containing severa l types of bacteria coming from different patients. The biofilm thickness when severa l types of colonies are present is higher than the thickness of monomicrobian colonies (Siebel et al. 1991) bi ofilms. Therefore experiments with the mucus containing different type of bacteria would be relevant. The data provided could be compared with the previous experiments run at the same bacteria concentration. Several parameters can influence the bacteria adhesion and will be modified to characterize their effect (Temperature, time of exposure, viscosity of the flow, flow rates ...). The mucus viscosity should be assessed for each experiment using magnetic rh eometry. The pressure of the cuff balloon is an important factor that should be characterized The protocol previously mentioned could be modified and the dry weight of biofilm could also be quantified. SEM analysis would be interesting in order to charac terize the biofilm structure. Mo reover, as the surface of the endotracheal tube influences the bacteria adhe sion, future work could be done improving the topographies of this surface. Topographies of the Endotracheal Tube The endotracheal tube surface is a parameter th at will be important in terms of bacteria adhesion. It has been showed by Chung et al. (2007) that diffe rent micro topographies based on the skin of sharks, Sharklet AFTM, can affect and decrease biofilm formation. For this study, Poly(dimethyl siloxane) elastomer was used to cr eate different surface topographies (Figure 7-1).

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61 Manufacturing different endotrach eal tubes with different patt erns and ran under the same condition will be relevant to diminish the adhesion. Figure 7-1. Micro-topographies fabricated in PDMSe from silicon wafer templates

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62 Conclusion The device that has been designed mimics bot h flows. The novelty of this device was to create a mucus flow that covered the wall of the artificial trachea. This objective was complete as it was proved with the experiments described chap ter 5. The protocol for testing endotracheal tube bacteria adhesion was set up so that future experiments will allow a better understanding of the adhesion process. The egg white experiment has shown that the bacteria built biofilm in the artificial trachea. The air dried the egg white as the mucus dries in the trachea of diseased patients. The bacteria growth was low compared to the growth that usually occurs in a diseased trachea because of the air flow system us ed. The model of trachea designed mimics physiologically the conditions of a diseased patient. Several expe riments could be carried out using this tracheal flow chamber. The bacteria and mi crobioal adhesion fields are wide and still needs experiments to better unders tand the process of adherence. The final objective will be to diminish the biofilm growth and minimize the infections in hospitals. As million of people are intubated every day, the improvement of the surf ace of endotracheal tube s will represent a real break-through in the medical and microbiology fields.

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63 APPENDIX A INTUBATION, DISEASED EN VIRONME NT AND BACTERIA Pseudomonas Aeruginosa Figure A-1. Schematic of bacteria Figure A-2. Pseudomonas aeruginosa infection of human respirator y mucosa in an organ culture caused patchy epithelial damage after 8 h. P. aeruginosa adhered to mucus and damaged epithelium, but not to normal epithelium. (Scale bar = 2.67 m). (Wilson et al. 1987)

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64 Intubation Twenty-eight percent of patients receiving mechanical ventilation will continue to have complication of ventilator-associated pneumonia (VAP) Mortality ranges : 28% to 70% The ventilator tubing is an im portant source of contamination As Pseudomonas Aeruginosa are hard to eradicate by antibiotic mean the solution would be to minimize the bacterial ad hesion on the endotracheal tube. Figure A-3. Endotracheal tube after being placed th rough the vocal cords and keeping them open.

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65 APPENDIX B FLOW CHAMBER DESIGN The parallel plate flow cham ber is one of the most common designs in terms of adhesion test under flow conditions (Figure B-1). More accurate than any other static method this design provides several advantages. Ind eed, it is convenient in terms of assessing the bacteria adhesion rate under a flow. This design a llows the microscope observation and can test different material. Different patterns of Sharklet can be tested in mucus environment or an airflow environment. This device is particularly us eful if one wants to understand mo re about the adhesion process, following the evolution of the biof ilm growth over several days a nd bacteria properties such as zeta potential. Figure B-1. Design of a para llel-plate flow chamber PTFE Gasket that designs the flow chamber Microscope observation Microscope slide Sample Bottom plate that will regulate the sample temperature Base Flow

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66 Mucus Flow Lets calculate the maximum flow rate of the mucus, knowing that the minimum flow rate is close to zero L/s (quasi-static). We can a ssess the maximum flow rate if we think of the problem in its higher conditions. We will then consid er that the diameter of the trachea is 2.5 cm and that the mucus thickness is maximum (273 m). We adopted for th e calculus the cylindershaped model of the trachea (Figure B-2). Figure B-2. Cylinder-shaped model of the trachea Reynolds Number The Reynolds number can be easily calcula ted knowing the flow rate the fluid characteristic and the main dimension of the flow chamber. Re mucus = ( .Qpp )/ (h0. ) (B-1) : fluid density (kg per cubic meter) Qpp: the volumetric flow rate (in cubic meters per second) h0: the distance between the parallel plates (meters) : the absolute viscosity Lets consider a flow chamber with th e following dimensions (Figure B-3) w0 = 20 mm h0 = 1 mm L0 = 30 mm Trachea Mucus layer

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67 Figure B-3. Dimensions of the flow chamber (l0, w0 and h0) and distances in the different directions (l, w, h) Calculus of the Mucus Flow Characteristics The trachea diameter is considered as equal to 2.5 centimeters and the mucus thickness of 273 micrometers. /4.(Dt)/4.(Dt-Lm) = 1.0662151 e-5 m (B-2) The maximum flow rate of the mucus will be assessed for the maximum speed, which means, Vmax = 35mm/min in the case of a healthy person. Qmax mucus = 6.21959 L/s (B-3) Re mucus = ( .Qmax mucus )/ (Lm. ) (B-4) The design of the experiment will have larger dimensions so lets use the concept of the Similitudes to assess the flow rate in our flow chamber. The dimensions of the parallel-plates flow chamber are 1 x 20 x 30 mm (ho x wo x Lo). Knowing that the developed profile is established when the laminar regime is set up: for a rectangular channel this length is equal to 0.09.Re.h = Le.

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68 Size a Parallel-Plate Flow Chamber The section of the channel has be en chosen rectangular as the sample has to be placed in the cell. The channel could also be cylindrical but it leads to optical difficulties later on if a microscope observation is required (the image would be distorted). The first important parameters to design a flow chamber are the hydrodynamic constraints. In order to get a laminar flow (Re < 2000) the Re ynolds number has to be chosen inferior to 100. As the mucus flow is naturally laminar this will be the case. On the other hand if the sample has to be tested under airflow conditions the regime being turbulent this design might affect the results. In a laminar flow the observation has to be done at the point wher e the profile is fully developed which means where L > Le, where Le is the length where the developed profile is fully established. Moreover, all calculations co nsidering a rectangular se ction assume that the height is much smaller than the width. This condition is set by the following equation: 2h/w<0.1 where h is the height of the channel and w its width. Similitudes The similitudes keep the Reynolds number constant. Re mucus real = Re flow chamber model (B-5) Considering that the properties of the mucus ar e close to the mucus simulant we will use in our model, we will consider that the density and the viscosity are equals. Then the equation above can be written Qmax mucus / Lm = Qmax model / ho (B-6) The maximum flow rate in the flow chamber can be determined. Qmax model = ho/Lm. (Qmax mucus) (B-7) Qmax model = 22.7 L/s (B-8)

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69 This maximum flow rate represents the flow rate in the flow chamber that will be used if a healthy environment is modelled. In the case of an intubated patient, the flow rate will be much smaller. As the bacteria adhesion is directly a ffected by shear forces a flow chamber experiment gives more data on the rate of biofilm growth unde r laminar flow conditions. It is an interesting experiment in terms of testing surface samples showing different topologies. The tracheal model chosen is closer to the real environment. However, it will give information after a certain time of incubation where this process allows following the evolution over the time the experiment is carried out. The flow chamber could then be a complementary experiment in order to understand better the adhesion process of the tested bacteria.

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70 APPENDIX C JOURNAL OF DRAWINGS

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83 APPENDIX D FLOW CHARACTERIZATION Figure D-1. Initial fl ow: Time 0:00 to 5:30

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84 Figure D-2. Flow evolut ion: Time 7:00 to 14:00

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85 Figure D-3. Final flow covering the entire surfac e of the lexan tube: Time 15:00 to 25:00

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86 APPENDIX E EXPERIMENTAL PROTOCOL FOR BACTERIA ADHESION TESTS Objective To assess the bacterial growth on endotracheal tube Kimberly-Clark adult size in the tracheal flow chamber that was designed. Material List Tracheal flow chamber and tubings One air filter Microcuff endotracheal tube Kimberly-Clark, oral/n asal Magill, Murphy Eye Air Cadet Cole-Parmer pressure-vacuum station Two 200ml beakers One 300mL beaker Four small glass cups Two glass cups Two Allen wrenches 3/32, 5/32 One pair of scissors One hemostat Three 30mL syringes Organism: Pseudomonas Aeruginosa Egg white to mimic the mucus Tryptic soy broth 18x150 mm sterile culture tubes with screw caps 500mL sterile baffled flask Self-incubated (waterbath) rotary shaker (New Brunswick Scientific Model C-76) Sterile Pasteur pipettes and a vacuum aspirating Unico 1100 Spectrophotometer for optical absorbance measurements 70% ethanol sterile wipers Sterile gloves Disassembly of the Tracheal Flow Chamber The connectors and the tubes have to be di sconnected of the syringe and the device. Figure E-1. Tubings and connector, mucus entrance

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87 Figure E-2. Tracheal flow chamber and the corresponding part numbers The parts 5 and 8 are not manufactured yet. The le xan tube (part 7) is directly connected to the top cover (part 3) and the cylindrical bottom (part 10). Unscrew the lexan tube (part 7) from the top cover (part 3) and the cylindrical bottom (part 10). Unscrew the mucus top tank (part 4) from the top cover (part 3). Remove the 3 drilled screws (part 1). Unscrew part 2. Remove the o-rings from the top cover (part 3) and the mucus tank (part 4).

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88 Figure E-3. Parts 1, 2, 3, 4 disassembled Remove the cover from the mucus reservoir (part 9). Unscrew the reservoir (part 11) from the base (part 14). Figure E-4. Mucus reservoir di sassembled (parts 9 and 11) Unscrew the feet from the base (part 14). Remove the larger o-ring from its groove. Figure E-5. Feet disassembled On the bottom of the base (part 14) unscrew the cylindrical bottom (part 10). 11 9 2 4 1 3

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89 Unscrew the fitting balloon (part 13) from the cylindrical bottom (part 10). Figure E-6. Cylindrical bottom (part 10) and fitting balloon (part 13) Figure E-7. Device disassembled 13 10 7 3 9 14 13 10 11 4 2 1 12 18 16 17

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90 Autoclave Procedure Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable systems available for the decontamination of labor atory waste and the ster ilization of laboratory glassware, media, and reagents. Material Preparation Wear closed toed shoes. Wrap parts 9, 10, 11, 14 in aluminum foils. Put parts 3, 4 and 10 in autoclave bags. Wrap two 200mL beakers, one 300mL beaker, 4 small and 2 big cups with aluminum foils. Put every part on the tray. Put autoclave tape on every part. Put the tray on the table; bring heat-i nsulating gloves to the autoclave room. Loading Autoclave Check that the chamber pressure is zero or that the cycle is complete. Stand behind door and slowly open it. Put the tray inside it. When closing the door, pull it toward you and th en push to close it in order not to let the steam go out. Operating Autoclave Push twice the button Change values. In the option menu, change cycle number 1 to a gravity cycle. Choose 15min (STER) as the duration of the cycle. Choose TEMP=250F as the temperature. Choose the drying method: DRY=30min. Save the values. Choose program 1 (program just set up), then push it a second time to start the autoclave cycle. Stay in the room to check that the door was properly closed and no steam comes out. Fill the autoclave book. Unloading Autoclave Wear heat-insulating gloves and closed toed shoes. Make sure the cycle is complete. Stand back from the door as a precaution and open it slowly. Remove the tray from autoclave and put it on the table. Check if every part has been correctly autoclaved (the autoclave tape and the autoclave bags must have changed aspect). Keep gloves on until the table is brought back to the lab.

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91 Place hot items in area that clear ly indicates the items are hot until they cool down to room temperature. Figure E-8. Autoclave parts Sterilization and Preparation of the Material The biological hood is cleaned with sterile et hanol wipes before starting. The hemostat (part 18), the Allen wrenches 3/32 and 5/32 in (p arts 16 and 17), and scissors are bleached then rinsed with 70% ethanol and air dried under the hood. The o-rings are made of buna and cannot w ithstand such high temperature (up to 120C/248F). Since the autoclave is done at 250F it is safer to not autoclave them. The screws, the fitting balloon (part 13), the airflow entrance screw (part 2), th e 3 drilled screws (part 1) are put in a cup filled of 70% ethanol for 10 min and later stirred. The parts are removed with the hemostat and place them in a steril e cup to air dry them under the hood. The tubings are rinsed out with bleach then with 70% ethanol (later sterile tubes will be used for the experiment). When the adaptive part s are available (parts 5 and 8), the lexan tube could be autoclaved or disposed. For this first experiment it is bleached, rinsed with 70% ethanol and air dried under the hood. The balloon is filled with bleach and rinsed wi th 70% ethanol and air dried. The outside of the air filter is cl eaned with 70% ethanol and air dried.

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92 Marks with a Sharpie pen are made on the endot racheal tube to cut 1 centimeter rings at the end of the experiment. The segments are 1 centimeter length and 5 millimeters apart (Figure E-9). A tube is attached to the endotracheal tube and allows the cu ff balloon to be inflated. At the extremity of this tube, the valve is cut and repl aced by a plastic luer (glue it). The endotracheal tube is rinsed with 70% etha nol and air drie d under the hood. Figure E-9. Endotracheal tube segments marks Assembly of the Tracheal Flow Chamber under the Biological Hood The device has to be assembled under the biological hood weari ng sterile gloves. Start assembling the lower part (parts 9 to 14). Do not connect the lexan tube (part 7) to the lower part. Assemble parts 1,2,3,4. Insert the endotracheal tube (part 6). Connect the lexan tube to the top cover (part 3). Pull out the tube and plastic luer used to inflate the cuff balloon through the hole on the top of the lexan tube. For that purpose use the hemostat. Egg Whites Preparation The eggs should not be older th an a week and should be kept refrigerated at 5C.The eggs are rinsed under tap water and air dried. The eggs are prepared following the method below: Wear sterile gloves under the hood. Wrap eggs with sterile ethanol wipes. Crack 6 eggs in a sterile small glass cup and separate the yolk (put the yolk in another cup with the shells).

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93 Figure E-10. Egg white separation Pour the cup in a sterile 200mL beaker (this is the coating). Crack 6 eggs in the cup and pour the content in another 200mL beaker (this is the culture media). Culture Preparation Twenty four hours prior to th e experiment, place one isolate Pseudomonas Aeruginosa in 5mL triptic soy broth (TSB) media in a 18x150 mm sterile culture tube with screw cap. The culture tube is placed in a waterbath/shaker at 37C a nd 150 rpm for overnight growth. Remove the tube prepared previ ously from the waterbath/shaker. Pipette 0.5mL of TSB solution containing P. Aeruginosa in 100mL of TSB media into a 500mL sterile baffled flask. Place the 500mL baffled flask containing the bacterial suspension in a waterbath/shaker at 37C and 150rpm. Check the optical absorbance with the Un ico 1100 spectrophotometer at 30 minutes intervals until the reading can be related to 106 CFU/mL by the two equations relating optical density to growth curves of P. Aeruginosa. 10mL of the bacterial suspension are inserted in the 30mL syringe. 20mL of egg white are inserted in the same syringe. The syringe is placed in the waterbath/sh aker at 37C and 150 rpm to homogenize the content. Coating Method As it was shown chapter 5, to obtain a better homogeneous flow the lexan tube has to be coated following the procedure below. Fill a 30mL syringe with egg white. Connect the tubes to the syringe a nd the 3 drilled screws (part 1). Place the assembly composed of the lexan tube, the endotracheal tube and the parts 1, 2, 3 and 4 above the 300mL beaker.

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94 Insert 15mL of the syringe in the lexan tube. The other extremity of the lexan tube is opene d. Plug the hole on the side with your finger. Place the lexan tube upside down and pour directly the content of the beaker into it (about half of the lexan tube is fill ed). Homogenize the coating. Put the lexan tube in the 300mL beak er to let the coating flow down. Figure E-11. Coating operation Put the 200mL beaker with the rest of egg white used for the coating, under the device (between the 4 feet). Connect the lexan tube to the lower part of the device. When there is no leakage through the fitting ba lloon (part 13), remove the beaker and plug the balloon. Clean the outside of the device with ethanol wipes. Insert the air filter at the top of the endotracheal tube. Operating Procedure The flow chamber has been assembled under the hood in a sterile way. The syringe pump is in the incubator, and the flow rate has to be set at 20L/min. Set the temperature of the incuba tor at 37C (body temperature). Connect the tube and syringe to the lu er and inflate the cuff (15mL of air). Tape the syringe to keep a cons tant pressure in the balloon. Set up the syringe containing the egg white and the bacteria on the syringe pump. Place the flow chamber in the incubator. Hole on the lexan tube to plug Extremity where the content of the beaker is poured directly 30mL syringe filled with egg white 200 mL beaker with 6 egg whites used for the coating

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95 Connect the tube from the Air Cadet Cole Parm er Pressure Vacuum Station to the filter. Disconnect the sterile syringe containing the coating and connect the syringe containing bacteria. Press start on the Air Cadet and make sure th e balloon (breathing bag) does not burst or is properly connected. Press start on the syringe pump. The experiment is run for 24 hours. Figure E-12. Experimental set up Bacteria Count After 24 hours of incubation, the tracheal flow chamber is removed from the incubator and placed under the hood. Wearing sterile gloves, the top cover (part 3) is slowly disconnected from the lexan tube without touching the endotracheal tube. Holding th e endotracheal tube with the hemostat and the top cover with the left hand the endotracheal tube is pulle d out of the stainless steel parts (Figure E-13).

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96 Figure E-13. Extraction of the endotracheal tube Six 1 cm-rings of the endotracheal tube are cu t (two at the cuff level, three above and another at the tip). The e ndotracheal tube is hold at the extremity with the hemostat and segments are cut with scissors (F igure E-14) without alteri ng the biofilm (the scissors is cleaned with a sterile 70% ethanol wi pe after each cut). Figure E-14. Cutting method The ETT segments are rinsed in PBS. The bi ofilm is homogenized by vortex mixing 1-cm rings of tubing or cuff material in a biofilm surfactant solution (Medtronic Xomed Inc.). Samples (100 L) of 6-fold dilutions of homogenized biofilm are each spread on two 5% horse blood agar plates. Viable counts are made by serial dilu tion-agar plate technique (Figure E-15) after overnight incubation in air at 37C.

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97 Figure E-15. The standard plate count tech nique to determine the total number of microorganisms in a sample (Aneja 2005)

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98 LIST OF REFERENCES [1] http://cfri.org/framesfaq2.htm Adair, C.G, S.P. Gor man, B.M. Feron, L.M. Byers, D.S. Jones, C.E. Goldsmith, J.E. Moore, J.R. Kerr, M.D. Curran, G. Hogg, C.H. Webb, G.J. McCarthy and K.R. Milligan. 1999. Implications of endotracheal tube biofilm for ventilator-associated pneumonia. Intensive Care Med, 25: 10721076. Afzelius, B. A. and B. Mossberf. 1980. Immobile cilia. Thorax, 35: 401-404. Allison, D.G. 1993. Biofilm-associated exopolysaccharides. Microbiology Europe, Nov./Dec: 16-19. An, Y.H. and R.J. Friedman. 1998. Concise Review of Mechanisms of Bacterial Adhesion to Biomaterial Surfaces. John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater), 43: 338 348. Aneja, K.R. 2005. Experiments in Microbiology, Plant Pathology and Bi otechnology. New Age Publisher Agarwal, M., M. King, and J.B.Shukla. 1994. Mucous gel transport in a simulated cough machine: effects of longitudina l grooves representing spacings between arrays of cilia. Biorheology, 31: 11. Bellon-Fontaine, M-N. and R. Briandet. 2000. Le Biofilm, une stratgie de survie pour les microbes, Salles propres & matrise de la contamination 9: 46-56. Bryer, J.D. 1987. Biologically active surfaces: process governing the formation and persistance of biofilms. Biotechnology Progress, 3: 57-68. Carpentier, B. and O. Cerf. 1993. Biofilms and their consequences, w ith their particular reference to hygiene in the food industry. Journal of Applied Bacteriology, 75: 499-511. Cerca, N., G.B. Pier, R. Oliveira and J. Azeredo. 2004. Comparative evaluation of coagulasenegative staphylococci (CoNS) a dherence to acrylic by static method and a parallel-plate flow dynamic method. Research in Microbiology, 155: 755-760. Chung, K. K., J.F. Schumacher, E.M. Sampson, R.A. Burne and P.J. Antonelli,. 2007. Impact of engineered surface microtopography on biofilm formation of Staphylococcus aureus. Biointerphases Vol. 2, Issue 2: 89-94. Cook, D.J., S. D. Walter, J. R. Cook, L. E. Griff ith, G.H. Guyatt, D. Leasa, R.Z. Jaeschke, and C. Brun-Buisson. 1998. Incidence of and Risk Factors for Ventilator-Associated Pneumonia in Critically Ill Patient. Annals of Internal Medicine 15 Sept., Vol. 129, Issue 6: 433-440.

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99 Costerton, J.W., K.J. Cheng and G.G. Geesey. 1987. Bacterial biofilms in nature and disease. Annual Reviews of Microbiology, 41: 435-464. Crouch Brewer, S., R.G. Wunderink, C.B. Jones and K.V. Leeper. 1996. Ventilator-associated pneumonia due to pseudomonas aeruginosa Chest, 109:1019-1029. De Bentzmann, S., P. Roget and E. Puchelle. 1996. Pseudomonas Aeruginosa adherence to remodelling respiratory epithelium. European Respiratory Journal, 9: 2145-2150. De Queiroz Guimares, M.M. and J.R. Rocco. 2006. Prevalence of ventilator-associated pneumonia in a university hospital and prognosis for the patients affected. J Bras Pneumol. 32(4): 339-46. Dickinson, R.B. and S.L. Cooper. 1995. Analysis of shear-dependent bacterial adhesion kinetics to biomaterial surfaces. Bioeng Food Nat Prod, 41: 2160-2174. Drenkard, E. and F.M. Ausubel. 2002. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation, Letters to Nature, Nature, 18 april,Vol 416. Duddridge, J.E., C.A. Kent and J.F. Laws. 1982. Effect of surface shear stress on the attachment of Pseudomonas fluorescens to stainless steel unde r defined flow conditions. Biotechnol Bioeng 24: 153-164. Duguid, I.G.,E. Evans, M.R. Brown and P. Gilb ert. 1992. Effect of bi ofilm culture upon the susceptibility of Staphylococcus epidermidis to tabramycin. J Antimicrob Chemothe, 30: 803810. Dullenkopf, A., A. Gerber and M. Weiss. 2003. Flui d leakage past tracheal tube cuffs: evaluation of the new Microcu ff endotracheal tube. Intensive Care Med, 29: 1849-1853. George, D.L, P.S. Falk, R.G. Wunderink, K.V. L eeper, G.U.Jr. Meduri, E.L. Steere, C.E. Corbet and C.G. Mayhall. 1998. Epidemiology of ventil ator-acquired Pneumonia based on protected bronchoscopic sampling. Am J Resp Crit Care Med Vol 158, 1839-1847. Golding, I., Y. Kozlovsky, I. Cohen and E. Be n-Jacob. 1998. Studies of Bacterial Branching Growth using Reaction-Diffusion Models for Colonial Development. Physica A Vol. 260, Num ber 3, 15 November: 510-554. Guyton, D., M.J. Banner and R.R. Kirby. 1991. High-volume, low-pressure cuffs, Are they always low pressure?. Chest, 100:1076-1081. Hoiby, N. 1982. Microbiology of lung inf ections in cystic fibrosis patients. Acta Paediatr. Scand. 301 (Suppl.): 33-54. Inglis, T.J.J., Lim E.W., Lee G.S.H., Cheong K.F. and Ng K.S. 1998. Endogenous source of bacteria in tracheal tube and proximal ventilator breathing system in intensive care patients, British Journal of Anaesthesia 80: 41-45.

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100 Inglis, T.J.J., M.R Millar., J.G Jones and D.A.Robi nson. 1989. Tracheal Tube biofilm as a source of bacterial colonization of the lung. Journal of Clinical microbiology, Sept: 2014-2018. Katsikogianni, M. and Y.F. Missirlis. 2004. Phys ico-chemistry of initial microbial adhesive interactions its mechanisms and methods for study. European Cells and Materials Vol. 8: 3757. Jayaraman, S., N. Soo Joo, B. Reitz, J.J. Wine and A. S. Verkman. 2001. Submucosal gland secretions in airways from cystic fibrosis pa tients have normal [Na+] and pH but elevated viscosity.P nas Vol. 98.No. 4: 8119-8123. Kieser-Nielsen. 1953. Mucin. Dansk Videnskabs Forlaf, Copenhagen. Klapper, I., C.J. Rupp, R. Cargo, B. Purvedorj and P. Stoodley. 2002. Viscoelastic fluid description of bacterial biofilm material properties. Biotech Bioeng 80: 289-296. Konrad, F., R. Schiener, T. Marx and M. Georgieff. 1995. Ultrastructure and mucociliary transport of bronchial respiratory epithelium in intubated patients. Intensive Care Med 21: 482489. Koerner R.J. 1996. Contribution of endotracheal tubes to the pathogenesis of ventilatorassociated pneumonia. Journal of Hospital Infection 35: 83-89. Korber, D.R., R. Lawrence, H.M. Lappin-Scot t and J.W. Costerton. 1995. Growth of microorganisms on surfaces. Microbiol Biofilms Edited by H. M. Lappin-Scott and J.W. Costerton, Cambridge University Press Kraigsley, A., P. D. Ronney and S. E. Finkel, Hydrodynamic influences on biofilm formation and growth, http://carambola.usc.edu/resear ch/biophysics/B iofilms4Web.html Lambert, P.A. 2002. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa Journal of the Royal Society of Medicine Suppl. No. 41, Vol. 95: 22-26. Marshall, K.C. 1985. Mechanisms of bacterial ad hesion at solid-water interfaces. D.C. Fletcher M., Eds Bacterial adhesion. Mechanisms and Physiology Significance. New York: Plenum Press : 133-161. Morgan, L., M. Pearson, R. de Iongh, D. Mackey H. van der Wall, M. Peters and J. Rutland 2004. Scintigraphic measurement of tracheal mucus velocity in vivo. Eur Respir J, 23: 518. Morton, I.H.G., D.L.A. Greenway, C.C. Gaylar de and S.B. Surman. 1998. Consideration of some implications of the resist ance of biofilms to biocides. International Biodeterioration and Biodegradation, 41: 247-259.

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101 Pucelle, E., O. Bajolet and M. Ably. 2002. Airway mucus in Cystic Fibrosis, Paediatric Resp. Reviews 3: 115-119. Rijnaarts, H.H.M., W. Norde, E. J. Bouwer, J. Lyklema and A. J. B. Zehnder. 1993. Bacterial Adhesion under Static and Dynamic Conditions. Applied and Environmental Microbiology Oct., Vol. 59, No. 10: 3255-3265. Roosjen, A., H.J. Kaper, H.C. Van der Mei, W. Norde and H. J. Busscher. 2003. Inhibition of adhesion of yeasts and bacteria by poly(ethylen e oxide)-brushes on glass in a parallel flow chamber, Microbiology 149: 3239-3246. Rumbak, M.J. 2002. The Pathogenis of Ventilator-Associated Pneumonia. Seminars in Respiratory and Critical Care Medicine volume 23. Ryan, K.J and C.G. Ray. 2004. Sherris Medical Microbiology 4th ed., McGraw Hill. Schlesinger, R.B. 1973. Mucocilia ry Interaction in the Tracheobr onchial Tree and Environmental Pollution. BioScience Vol. 23, No. 10 (0ct): 567-573. Scott, H. Randell and R.C. Boucher. 2006. Effectiv e mucus clearance is essential for respiratory health. Am J Respir Cell Mol Biol Vol 35: 20-28. Siebel, M.A. and W.G. Characklis. 1991. Ob servation of binary population biofilms. Biotech. Bioeng, 37:778-789. Sims, D.E., and M.M. Horne. 1997. Heterogeneity of the compositi on and thickness of tracheal mucus in rats. The American Physiology Society, 1036-1041. Song, L.F., P.R. Johnson and M. Elimelech. 1994. Kinetics of colloid deposition onto heterogeneously charged surfaces in porous media. Environ. Sci. Technol, 28: 1164-1171. Stewart, P.S. and J.W. Costerton. 2001. Antibio tic resistance of bacteria in biofilms. Review. The Lancet, July 14, Vol 358. Stoodley, P., Z. Lewandowski, J.D. Boyle and H .M. Lappin-Scott. 1999. Structural deformation of bacterial biofilm caused by short-term fluctuati ons in fluid shear: an in situ investigation of biofilm rheology, Biotechnol Bioeng, 65: 83-92. Taboada-Serrano, P., V. Vithayaveroj, S. Yiacoumi and C. Tsouris. 2005. Surface charge heterogeneities measured by atomic force microscopy. Environ. Sci. Technol., 39: 6352-6360. Terada A., A. Yusasa, T. Kushimoto, S. Tsuneda, A. Katakai and M. Tamada. 2006. Bacterial Adhesion to and viability on positively charged poylmer surfaces. Microbiology, 152: 35753583.

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102 Tippe A., R. Korbel, A. Ziesenis and J. Heyder. 1998. Viscoelastic properties of canine tracheal mucus: effect of structural inhomogeneities and storage. Scand J Clin Lab Invest 58:529-264. Tomkiewicz, R. P., G. M. Albers, G. T. De Sanc tis, O. E., Ramirez, M. King, and B. K. Rubin. 1995. Species differences in th e physical and transport proper ties of airway secretions. Can. J.Physiol. Pharmacol 73: 165. VadilloRodriguez, V. and E. Logan. 2006. Localized Attraction Correl ates with Bacterial Adhesion to Glass and Metal Oxide Substrata. Environ. Sci. Technol. 40: 2983-2988. Van Loosdrecht, M. C. M., L. Lyklema, W. Nord e and A.J.B. Zehnder. 1989. Bacterial adhesion: a physicochemical approach. Microb. Ecol ., 17: 1-15. Wanner A. 1977. Clinical aspect s of mucociliary transport. Am Rev. Respir. Dis., 116: 73-125. Weiss M., G. Shorten, K. Stutz and V. Bernet 2007. Tracheal Sealing by auto-inflation in tracheal tube cuffs. Pediat ric Anesthesia, 17: 243. Wilson R., R.B. Dowling and A.D. Jackson. 199 6. The Biology of bacterial colonization and invasion of the respirator mucosa. European Respiratory Journal, 9: 1523-1530. Wilson, R., T. Pitt, G. Taylor, D. Watson, J. MacDerot, D. Sykes, D. Roberts and P. Cole. 1987. Pyocyanin and 1-Hydroxyphenazine Produced by Pseudomonas aeruginosa Inhibit the beat of Human respiratory Cilia in vitro. J. Clin. Invest., 79, January : 221-229. Yadav, N.K., D.V. Vadehra. 1977. Mechanism of eff white resistance to bacterial growth. Journal of Food of Science, 42: 97-99. Young, P.J., M. Rollinson, G. Downward, S. Henders on. 1997. Leakage of fluid past the tracheal tube cuff in a benchtop model. British Journal of Anaesthesia 78: 557-562.

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103 BIOGRAPHICAL SKETCH Karine Vizcaino obtained a scien tific baccalau reate with distinction from the French school Lyce Jules Verne in 2002. For 2 years she prepared the national competitive exam to enter French Colleges of Engin eering (Classes Prparatoires a ux Grandes Ecoles) at the Lyce Condorcet (Paris IXe). She obtained a DEUG (General Degree from Jussieu University (Paris)) of Science parallel to her pr eparation and entered the ENSAM (Ecole Nationale Suprieure dArts et Mtiers) in 2002, a well-known French college of engineering specializing in mechanical and industrial engineering and manuf acturing processes (ParisTech Group). In 2006, she decided to go abroad to finish her studies and applied for a double-degree. She joined the department of mechanical and aerospace engine ering and will receive her French Master of Engineering at the same time as he MS from the University of Florida. In 2007, she started to work as a Reaching Assistant for Dr. Tran-Son-Ta y who introduced her to the biomedical field. She decided to apply her mechani cal design and fluid mechanics skills to the design of a tracheal flow chamber for testing endotra cheal tube bacteria adhesion.