University of Iowa

Iowa Research Online

Theses and Dissertations

Spring 2015

Thermal deactivation of Pseudomonas aeruginosa

biofilms
Ann Marie O'Toole
University of Iowa

Copyright 2015 Ann O'Toole

This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/1715

Recommended Citation
O'Toole, Ann Marie. "Thermal deactivation of Pseudomonas aeruginosa biofilms." MS (Master of Science) thesis, University of Iowa,
2015.
http://ir.uiowa.edu/etd/1715.

Follow this and additional works at: http://ir.uiowa.edu/etd

Part of the Chemical Engineering Commons

THERMAL DEACTIVATION OF PSEUDOMONAS AERUGINOSA BIOFILMS

by

Ann Marie OToole

A thesis submitted in partial fulfillment

of the requirements for the Master of Science
degree in Chemical and Biochemical Engineering in the
Graduate College of
The University of Iowa

May 2015

Thesis Supervisor: Assistant Professor Eric Nuxoll

 Copyright by

ANN MARIE OTOOLE

2015

All Rights Reserved

 Graduate College
The University of Iowa
Iowa City, Iowa

CERTIFICATE OF APPROVAL

____________________________

MASTERS THESIS

_________________

This is to certify that the Masters thesis of

Ann Marie OToole

has been approved by the Examining Committee for

the thesis requirement for the Master of Science degree
in Chemical and Biochemical Engineering at the May 2015 graduation.

Thesis Committee: ____________________________________________

Eric Nuxoll, Thesis Supervisor

____________________________________________
Allan Guymon

____________________________________________
Jennifer Fiegel
 ABSTRACT

Bacterial biofilm infection is a common (~ 2 to 4%) complication for recipients of

surgically implanted medical devices. Due to the tremendous increase in antibiotic resistance

when these bacteria enter the biofilm phenotype, present treatment requires explantation and

replacement of the device, often with multiple surgeries and always with much longer patient

recovery time. The specific objective of this study was to quantify the degree of deactivation of

biofilm from exposure to thermal shock for varying temperature and time durations. While

extreme temperature (>150C) is routinely used to sterilize (e.g. autoclaves), such temperatures

have a severe cost within the body. Despite extensive studies on thermal deactivation of bacteria

in the planktonic phenotype over a wide range of temperatures (e.g., pasteurization protocols),

surprisingly little is known about the thermal deactivation of biofilms except under extreme

conditions. Here, the deactivation of Pseudomonas aeruginosa biofilms is reported. These

biofilms were cultured in two ways, using a batch mode setup with an orbital shaker and with a

continuous drip-flow reactor (DFR). These two culturing techniques resulted in different biofilm

architecture and a bacteria concentration 350 times higher for the DFR biofilms. The DFR

biofilms, becoming the focus of the study, were cultured at 37C for 24 hours and subjected to

heat shocks on the range of 50C to 80C for durations of 1 to 30 minutes. Heat shocks were

delivered by immersion in thermostatted media for the prescribed time and the resulting

concentration of colony forming units (CFU/cm2) were quantified using direct enumeration. Up

to 6.6 orders of magnitude reduction in CFU concentration was observed, indicating that thermal

deactivation is a reasonable approach to biofilm mitigation. Integrating this approach with a

magnetic nanoparticle implant coating will result in an innovative treatment for implant infections

in situ without explantation or device replacement.

ii
 PUBLIC ABSTRACT

Bacterial biofilm infection is a common (~ 2 to 4%) complication for recipients of

surgically implanted medical devices. Due to the tremendous increase in antibiotic resistance

when these bacteria enter the biofilm phenotype, present treatment requires explantation and

replacement of the device, often with multiple surgeries and always with much longer patient

recovery time. The specific objective of this study was to quantify the degree of biofilm

deactivation from exposure to thermal shock for varying temperature and time durations. While

extreme temperature (>150C) is routinely used to sterilize (e.g. autoclaves), such temperatures

have a severe cost within the body. Despite extensive studies on thermal deactivation of bacteria

in the planktonic phenotype over a wide range of temperatures (e.g., pasteurization protocols),

surprisingly little is known about the thermal deactivation of biofilms except under extreme

conditions. Here, the deactivation of Pseudomonas aeruginosa biofilms is reported. These

biofilms were cultured at 37C for 24 hours in a drip-flow reactor and subjected to heat shocks on

the range of 50C to 80C for durations of 1 to 30 minutes. Heat shocks were delivered by

immersion in thermostatted media for the prescribed time and the resulting concentration of

colony forming units (CFU/cm2) were quantified using direct enumeration. Up to 6.6 orders of

magnitude reduction in CFU concentration was observed, indicating that thermal deactivation is a

reasonable approach to biofilm mitigation. Integrating this approach with a magnetic nanoparticle

implant coating will result in an innovative treatment for implant infections in situ without

explantation or device replacement.

iii
 TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................................... vi

LIST OF FIGURES ....................................................................................................................... vii

Introduction and background ........................................................................................................... 1

What is a biofilm? ........................................................................................................................ 1

Antibiotic resistance of biofilms .................................................................................................. 2

Biofilm treatment and prevention ................................................................................................ 4

Thermal sterilization .................................................................................................................... 5

Objectives .................................................................................. Error! Bookmark not defined.

Materials and methods ..................................................................................................................... 8

Organisms and media................................................................................................................... 8

Growth study.............................................................................................................................. 10

Biofilm characterization via Confocal Laser Scanning Microscopy ......................................... 10

Direct enumeration of bacteria................................................................................................... 11

Shaker biofilm cultures .............................................................................................................. 12

Drip flow reactor (DFR) biofilm cultures .................................................................................. 13

Heat shock system...................................................................................................................... 14

Quantification via direct enumeration........................................................................................ 15

Statistical analysis ...................................................................................................................... 16

Results............................................................................................................................................ 16

Growth study.............................................................................................................................. 16

Shaker biofilms .......................................................................................................................... 17

Drip flow reactor control biofilms ............................................................................................. 19

Biofilm characterization via CLSM ........................................................................................... 20

iv
 DFR Heat shock biofilms........................................................................................................... 23

Discussion ...................................................................................................................................... 24

Conclusions and future investigations ........................................................................................... 32

References...34

v
 LIST OF TABLES

Table 1 - Average log(CFU/cm2) standard deviation of DFR biofilms as a function of heat

shock temperature and duration ..................................................................................................... 23

vi
 LIST OF FIGURES

Figure 1 - General treatment schematic for long-term goal of an innovative therapy for biofilm
infected medical devices.. ................................................................................................................ 7
Figure 2 - General setup for shaker biofilm growth....................................................................... 13
Figure 3 - General setup for drip flow reactor (DFR) biofilms. ..................................................... 14
Figure 4 - General setup for heat shock system. Biofilms were transferred to heat shock system
via frosted microscope slides.. ....................................................................................................... 14
Figure 5 - Relationship between inoculum growth time (h) and CFU/mL .................................... 17
Figure 6 - Control shaker biofilm results.. ..................................................................................... 18
Figure 7 - Control results for DFR biofilms .................................................................................. 19
Figure 8 - CLSM control biofilms.. ............................................................................................... 21
Figure 9 Representative confocal micrographs of a DFR control biofilm at 37C for 30 minutes
(A) compared to a heat shocked DFR biofilm at 80C and 30 minutes (B) .................................. 22
Figure 10 Confocal micrographs of shaker biofilms heat shocked at 80C for 1, 15, and 30
minutes.. ......................................................................................................................................... 23
Figure 11 Average log(CFU/cm2) for each DFR biofilm heat shock time and temperature
combination ................................................................................................................................... 24
Figure 12 - Effect of heat shock duration (A) and temperature (B) on log(CFU/cm2).. ................ 27
Figure 13 - Linear correlations of log(CFU/cm2) vs. temperature for each heat shock duration. As
time is increased, slopes remain similar between heat shock durations......................................... 28
Figure 14 - Representative linear regressions for log(CFU/cm2) vs. time (A) and log(time) (B) for
60C. .............................................................................................................................................. 29
Figure 15 - Correlation of cell death temperature dependence to log(time) .................................. 30

vii
Introduction and background

Biofilm formation due to a nosocomial infection is a serious issue for implanted medical devices.

Many sterilization protocols have been put in place to help reduce the occurrence, but the

problem persists. The typical treatment of these infections is to have subsequent surgeries to

explant and replace the device, as well as administer antibiotics to mitigate the bacteria. However,

this approach is not eliminating the problem because infection occurrence persists and the number

of procedures continues to grow (Burke 2003, Darouiche 2004, Wisplinghoff, Bischoff et al.

2004, Ammerlaan, Harbarth et al. 2013). This results in increased recovery time and medical

expenses for the patient. Therefore, it is desirable to develop a new and innovative treatment for

biofilm deactivation.

What is a biofilm?

The biofilm phenotype is an aggregation of bacterial cells that stick on a surface, characterized by

its layer-like structure and surrounding polysaccharide matrix. It is differing from the free-

floating, planktonic phenotype. The biofilm architecture in general is heterogeneous throughout

and constantly changing as the biofilm colonizes and grows (Zoubos, Galanakos et al. 2012).

Biofilm formation is usually broken down into five stages of growth (Drenkard and Ausubel

2002, Hall-Stoodley, Costerton et al. 2004, Zoubos, Galanakos et al. 2012). The first stage is the

initial attachment of bacterial cells to the surface. Stage two is the accumulation of cells, as the

bacteria concentration starts to increase. Stage three is the initial secretion and development of the

polysaccharide matrix. Stage four is the maturation of the biofilm and its overall heterogeneous

architecture thickness and bacteria concentration varying throughout the colony. The final stage

is the re-release of bacteria as the biofilm continues to spread and colonize other surfaces.

During these growth stages and phenotypic switch, the bacteria are increasing their resistance to

many means of chemical treatments specifically antibiotics.

1
Biofilms colonize approximately 2 to 4% of the 3.6 million implanted devices annually in the

U.S. (Darouiche 2004). Treatment can cost anywhere from $15,000 to $50,000 per surgery,

totaling an extra $5 billion in annual medical expenses (Darouiche 2004). The patient has also

been shown to be more susceptible to a second infection after treating the original (Stewart and

Costerton 2001, Drenkard 2003). For example, 1.5-2.5% of total hip and knee replacements

develop joint infections upon primary implantation. After replacement, the rates increase to 3.2-

5.6% (Rohde, Burandt et al. 2007). The main reasons these infections are difficult to treat is

because of the biofilms antibiotic resistance which make it up to 1000-fold less susceptible to

antibiotics than planktonic cells (Nickel, Ruseska et al. 1985, Costerton, Lewandowski et al.

1995, Vinh and Embil 2005).

Antibiotic resistance of biofilms

A biofilms antibiotic resistance is attributed directly to the biofilm phenotype. This resistance

should not be confused with evolutionary antibiotic resistance seen from overuse of antibiotics

and the development of resistant genes in antibiotics (Levy 1998). While it is still not completely

understood why the biofilm phenotype shows increased antibiotic resistance, it is hypothesized

that the reasons are multifactorial (Costerton, Lewandowski et al. 1995, Drenkard 2003). These

factors include transport limitation, physiological gradients, development of persister cells,

changes in quorum sensing (QS) systems, triggering the stress response, and up-regulation of

multidrug efflux pumps (Drenkard and Ausubel 2002, Drenkard 2003).

The polysaccharide matrix of the biofilm plays a large role in the transport limitation of

antibiotics and biocides, as well as physiological necessities such as oxygen and nutrients. The

matrix not only adds a physical barrier, but also has been shown to react with various chemical

treatments and render them unreactive (Mah and O'Toole 2001, Imaizumi, Tran et al. 2003). This

depends on the type of antibiotic being administered and the different strains of bacteria

comprising the biofilm. For example, Pseudomonas aeruginosa biofilm has been shown to slow

2
delivery of aminoglycosides but not fluoroquinolones (Stewart 2002). The charge of the antibiotic

is also important since positively charged species will bind to the negatively charged matrix

therefore slowing diffusion (Stewart and Costerton 2001). Limitations of oxygen and nutrients are

also attributed to the biofilm matrix. Bacteria located on the outer layers of a biofilm have better

access to nutrients and therefore a higher metabolic rate than bacteria located near the center of a

biofilm. This lower metabolic activity results in these bacteria being in a dormant state and thus

not up-taking the antibiotics. Also certain antibiotics, such as penicillin, only target bacteria in a

growth stage (Stewart and Costerton 2001).

The development of persister cells can be a result of the biofilm phenotype change and QS

systems. QS is the cell-to-cell communication in biofilms as bacteria concentration increases on a

surface. It triggers signaling by N-acyl homoserine lactones (for gram-negative bacteria), which

diffuse across the cell membrane and bind to regulatory proteins within the cell (Kalia 2013).

This signaling can result in the formation of the biofilm matrix and persister cells. These cells

have been shown to continue to thrive through repeated and prolonged antibiotic exposure. It is

thought that they are able to accomplish this by altering their typical programmed cell death

(PCD) that is usually experienced when exposed to certain antibiotics. For example, it was shown

that P. aeruginosa biofilms when exposed to fluoroquinolones, results in a decreased viable

bacteria population of 1,000 to 100,000 fold, but continuous increase of the antibiotic

concentration indicates any viable cells remaining are completely resistant (Lewis 2000). When

they do not follow the normal PCD, they are able to resist antibiotics and continue the biofilms

colonization.

Biofilm bacteria also activate the general stress response during development. This is due to

environmental stresses, DNA damage, osmotic variations, oxidative changes, etc. (Drenkard

2003). The stress response causes higher expression of the sigma factor rpoS, which activates a

number of genes necessary to maintain cell viability during stationary phase (Xu, Franklin et al.

3
2001, Wang and Wood 2011). The signaling of this sigma factor enables protection from

environmental stressors, which would include antimicrobial agents.

A final contributing factor to antibiotic resistance is the up-regulation of multidrug efflux pumps.

Efflux pumps help stabilize the bacteria by pumping out toxins such as antimicrobial agents,

metabolites, and QS signal molecules such as N-acyl homoserine lactones (Pearson, Van Delden

et al. 1999, Aeschlimann 2003, Soto 2013). When these pumps are up-regulated, it is harder to

target antibiotics to the biofilm (Soto 2013). What is not fully understood is what causes the

different pump systems to turn on and off (Aeschlimann 2003, Piddock 2006).

In general, biofilms have been shown to be antibiotic resistant for many reasons. Further

complicating matters, the biofilm may host multiple species of pathogens, each with different

resistance characteristics (Watnick and Kolter 2000, Stoodley, Sauer et al. 2002). These biofilm-

specific resistance problems suggest an inherent limit on the effectiveness of current chemical

approaches to biofilm deactivation.

Biofilm treatment and prevention

Recent attempts for combatting biofilms include biocide loaded polymer device coatings, which

attempt to keep the bacteria from ever colonizing (Kingshott and Griesser 1999, Smith 2005, von

Eiff, Jansen et al. 2005, Pavithra and Doble 2008), quorum sensing inhibitors (Davies 1998,

Boles and Horswill 2008, Kalia 2013), electrical current (Blenkinsopp, Khoury et al. 1992, Van

Der Borden, Van Der Werf et al. 2004), extracorporeal shock waves (Gerdesmeyer, von Eiff et al.

2005), photodynamic therapy (Di Poto, Sbarra et al. 2009), and magnetic field exposure (without

added magnetic material) (Stewart, Wattanakaroon et al. 1999).

Polymer coatings on medical implants usually employ the addition of functional groups in order

to deter bacteria adhesion and accumulation from ever happening. A common example of a

functional group addition is poly(ethylene glycol) (PEG). While PEG has shown to decrease the

4
adhesion of proteins, it is not always successful at keeping bacteria from colonizing. It is also

easily oxidized leaving the material susceptible to colonization (Cheng, Zhang et al. 2007).

Therefore, biocompatibility of the material is an important consideration. Zwitterionic polymer

coatings have also been developed as alternatives to PEG coatings because of their broader

chemical diversity (Mi and Jiang 2014). They use a homogenous distribution of anionic and

cationic groups on a polymer backbone, typically methacrylate or acrylamide, resulting in an

ultra-hydrophilic and neutrally charged polymer. However, the polymer alone is not sufficient to

keep bacteria from colonizing so antibiotics have been added as leaving groups on the polymers

in order to increase anti-fouling efficacy (Mi and Jiang 2012). Different materials have been used

in various medical applications but there is not one that appears to be universally applicable for

biofilm prevention (Pavithra and Doble 2008).

QS system inhibitors have been a developing area of interest for biofilm prevention. The concept

is straightforward biofilm development would be prevented if cells were not allowed to

communicate with each other as population density increases. Issues arise, however, when

targeting QS signal receptors, as many strains have multiple receptors to inhibit (Kalia 2013).

Also, even after QS systems are interrupted, an accumulated population of bacteria is already

colonized on the surface, leading to a vulnerable starting state for treatment.

All of the other prevention methods mentioned above (electrical current, shock waves,

photodynamic therapy, and magnetic field exposure) rely on a complementary means of treatment

(such as antibiotics) to be successful. Plus none of these methods have shown to mitigate all

pathogens or be effective and easy to accomplish in vivo. Therefore, it would be more

encouraging to develop a high deactivating, sole means of treatment that could be readily and

safely performed in vivo.

Thermal sterilization

5
Thermal sterilization is a well-established concept and is based on the fact that every organism

has a ceiling temperature for survival. For example, typical pasteurization involves heating milk

to 72 C for 15s in order to kill bacteria, and thermal sterilization of common pathogens like P.

aeruginosa in its planktonic phenotype have been studied at 50 60 C (Hassani, lvarez et al.

2005, Hassani, Manas et al. 2007). Less common are studies of thermal sterilization of biofilms,

even in non-medical applications where arbitrarily high heating can be conveniently applied. One

group has developed a predictive model for heat inactivation of Listeria monocytogenes biofilms

on food processing equipment (Chmielewski and Frank 2004, Chmielewski and Frank 2006), and

another group has shown magnetic nanoparticles can heat biofilms (Park, Park et al. 2011).

Otherwise thermal sterilization studies have commonly been accompanied by chemical and

mechanical means. A study has not been conducted to quantify deactivation based on specific

temperature protocols alone. Within the human body, such indiscriminate methods such as

oxidizers and high-shear scrubbing are unadvised and simply heating the biofilm becomes a

challenge. For devices without large batteries and wireless telemetry, the heating energy must be

supplied from outside the body. Being able to remotely heat a biofilm, would result in an

innovative, less invasive treatment method. Determining the physiologically relevant heat shock

protocols (time and temperature combinations needed for deactivation) is the initial step to

display this proof of concept.

Objectives

The long-term goal of this research is to develop an innovative treatment for eliminating biofilm

infection via remote heat sterilization. This heat in the future will be supplied wirelessly by

coating the implanted device with a magnetic nanoparticle-loaded polymer coating and exposing

the device to an external magnetic field (Figure 1). The magnetic field will cause the particles to

heat-up and this will thermally deactivate the biofilm on the surface of the implant.

6
Figure 1 - General treatment schematic for long-term goal of an innovative therapy for biofilm infected
medical devices. The implanted device will be coated in a magnetic nanoparticle-loaded polymer
film. Once a biofilm colonizes the device, an alternating magnetic field can be externally exposed, causing
the nanoparticles to wirelessly heat the biofilm and result in deactivation.

To establish this treatment, it is necessary to first determine the combination of heat shock

temperature and duration necessary for the level of deactivation that mitigates the biofilm. This

research focuses on this task through the following objectives:

1. Develop biofilm culturing protocol.

2. Evaluate the potential of different biofilm architectures and bacteria loading using

confocal laser scanning microscopy and direct enumeration quantification, respectively.

3. Develop a heat shock process that minimizes thermal inertia and instantaneously applies

a thermal shock to the biofilm.

7
 4. Determine the heat shock time and temperature combinations needed to reach different

levels of log reduction of viable bacteria. This will lead to the ability to approximate the

extent of deactivation based on thermal protocol.

This project has the potential to help hundreds of thousands people annually that develop an

infection of their implanted medical device. The experiments will develop biofilms, deactivation

and quantification procedures, and ultimately result in an innovative treatment for biofilm-

infected implants.

Materials and methods

Biofilms were cultured by two different methods and then heat shocked for varying temperature

and time combinations, ranging from 50C-80C and durations from 1 to 30 minutes respectively.

Post heat shock, biofilms were quantified via direct enumeration and their log reductions were

calculated.

All experiments and preparation work were completed in a biosafety level 2 room and

precautions were taken when addressing cleaning and contamination. This includes the use of

appropriate personal protective equipment including eye protection, lab coat, and nitrile gloves

sterilized with 75% by volume ethanol in water. All organism isolation and quantification of

bacteria was completed in a biosafety cabinet. Lab waste such as pipette tips and incoluating

loops were single use pieces and autoclaved prior to trials. Waste was also autoclaved prior to

disposal. Media was also autoclaved in order to ensure sterility and only openly exposed in the

biosafety cabinet. Heat shocks were completed in a water bath system (see Heat shock system

section below for details) with deionized water and dishes were sealed with parafilm to avoid

contamination. All instrumentation was routinely and thoroughly cleaned with 10% by volume

bleach in water solution and 75% by volume ethanol in water.

Organisms and media

8
The pathogen Pseudomonas aeruginosa was chosen for this investigation. It is commonly

associated with nosocomial infections and often used in biofilm research (Drenkard and Ausubel

2002, Borriello, Werner et al. 2004, Pierce 2005, Gellatly and Hancock 2013). It is also shown to

be highly resistant to antibiotics when present as the biofilm phenotype (Drenkard and Ausubel

2002, Gellatly and Hancock 2013). Specifically, reference strain 16952 (American Type Culture

Collection, Manassas, VA) was used, which is PA01 Pseudomonas aeruginosa and non-mucoid.

It was isolated from frozen glycerol stock cultures and streaked on agar-filled plates (Difco

Nutrient Agar, Sparks, MD). The streaking was done in a biosafety cabinet and occurs in three

different streaks, using three sterile inoculating loops (VWR, Radnor, PA). Each streak pulls from

the last part of the previous streak in order to dilute the bacteria throughout each streaked section

of the agar plate leaving the third section the most dilute and able to grow individual colony

forming units (CFUs). After streaking, the plates were incubated for 24 hours at 37C, and then a

single colony was used to inoculate 5 mL of tryptic soy broth (TSB; BD Bacto, Sparks, MD) with

a sterile inoculating loop in a standard 15 mL test tube. Single colonies were used to best control

starting inoculum concentration for biofilm culturing as well as genetic variations due to bacteria

replication. The TSB was sterilized prior to inoculation at 121C for 15 minutes in an autoclave.

The 5 mL of inoculated TSB was incubated at 37C for 24 hours, which results in a bacterial

concentration of approximately 109 CFU/mL (see Growth study below for methods). This

inoculum was then used for biofilm culturing. All streaking waste was autoclaved prior to

disposal.

Glucose-enhanced media was used in the shaker biofilm cultures that resulted in biofilms with

less dense bacteria concentrations than biofilms grown using TSB (see Shaker biofilms in the

Results section). To make 500 mL of media (all ingredients from Fisher Scientific, Waltham,

MA), 4.3 g of potassium monophosphate, 2.7 g potassium phosphate dibasic, 0.024 g magnesium

sulfate, 0.00144 g ferrous sulfate heptahydrate, and 5.232 g MOPS free acid were dissolved in

9
500 mL of purified water. This solution was then vacuum filtered (VWR, Radnor, PA) to ensure

sterility.

Growth study

In order to verify that the inoculum was at a consistent concentration and in stationary phase

before starting biofilm culturing, a growth study was done on P. aeruginosa strain 16952. The

strain was isolated as previously described from the frozen glycerol stock and streaked on an

agar-filled plate in the biosafety cabinet. The plate was incubated for 24 hours at 37C, and then a

single colony was used to inoculate 5 mL TSB. The inoculated broth was kept sealed at 37C, 5%

CO2 and evaluated over a 36 hour period. Samples taken at various time points during the study

with sterile pipette tips were serially diluted with clean broth to an appropriate dilution factor

based on how long the sample had already been incubated and the expected CFU/mL. The

samples were quantified by both direct enumeration (see Direct enumeration of bacteria below)

and by ultraviolet-visible (UV-VIS) spectrophotometric scans. UV-VIS (Varian Cary 50, Walnut

Creek, CA) scans were done from 200 to 800 nm focusing on the optical density (OD) at 600

nm because of the broths yellow color and the common use of this wavelength in growth studies

and bacterial turbidity measurements. After quantification, the samples were autoclaved prior to

disposal.

Biofilm characterization via Confocal Laser Scanning Microscopy

Confocal Laser Scanning Microscopy (CLSM) was used to image biofilms in order to evaluate

thickness and architectural characteristics. CLSM is an imaging technique that utilizes the fact

that only light at a certain depth reaches the detector. Multiple images can be taken throughout a

sample as the focal plane is moved to different depths. Rendering these slices together creates an

overall three-dimensional image.

10
The biofilms were imaged by CLSM using fluorescent dyes. The fluorescent stains used for P.

aeruginosa are based on DNA binding and membrane integrity. The live bacteria are stained with

SYTO 9 which complexes with DNA, and when bound, causes the bacteria to fluoresce green.

SYTO 9 also stains dead bacteria, but if the bacteria has a disrupted membrane, another stain,

propidium iodide, is also allowed into the cell, quenching the green fluorescence and fluorescing

red itself. The dyes were from the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen,

Eugene, OR), which calls for a 1:1 ratio of Component A (SYTO 9 dye 3.34 mM) to Component

B (propidium iodide 20 mM) for the staining procedure.

The CLMS used was an upright Bio-Rad 1024 confocal microscope (Hercules, CA) at Central

Microscopy Research Facility at the University of Iowa with a 40x dip lens. The dip lens was

cleaned pre and post sample exposure using sterile cotton swabs and 75% by volume ethanol in

water. Krypton/argon mixed gas lasers of 488 and 568 nm excited the SYTO 9 and propidium

iodide dyes, respectively. The lasers were used sequentially to avoid any overlap in the emission

of fluorescent light from the stains. The microscope created an overall image based on the

average of four scans, a z-step of 1 m, 256 intensity levels, and 512x512 pixel count. All

parameters were set using the Bio-Rad software. The slices were then rendered together to create

a three-dimensional image of the biofilm using the Java-based, public domain image processor,

ImageJ (National Institutes of Health, Bethesda, MD). Imaged biofilms were disposed of by

autoclaving.

Direct enumeration of bacteria

Direct enumeration is a common means of quantifying bacteria by serially diluting a sample,

plating it, and then allowing CFUs to grow for counting. All enumeration was done in the

biosafety cabinet with single use, autoclaved pipette tips. Growth study samples were thoroughly

mixed by inverting ten times. Serial dilutions were set up by using 900 L of TSB in each test

tube and then adding 100 L of undiluted sample to the first tube. Pipetting up and down ten

11
times mixed the dilution, and then 100 L of the first tube was put into the second tube of 900 L

TSB. This process was repeated until all dilutions were completed. Each dilution reduces the

CFU concentration by a factor of 10. After diluting, a 100 L sample from each tube was spread

over an agar-filled petri dishes using 4 mm diameter glass beads (Biochemistry Stores at the

University of Iowa, Iowa City, IA). The glass beads were discarded after spreading each plate.

After spreading the sample, the CFUs were incubated for 24 hours at 37C. Then the CFU/mL for

the original sample were back calculated using the following equation:

CFU/mL = ( ) 10 /0.1 Equation 1

Shaker biofilm cultures

Shaker biofilms were grown using the 5 mL of inoculated TSB at a bacteria concentration of 109

CFU/mL. They were cultured in a 4-well polystyrene rectangular dish (Thermo Scientific Nunc,

Rochester, NY) with a frosted glass microscope slide on the bottom of each well (Leica, Buffalo

Grove, IL). Glucose-enhanced media was added to the well along with inoculum in a 5:0.33 mL

ratio, respectively. The dishes were then sealed with parafilm (Bemis Company, Inc., St. Louis,

MO) and placed on an orbital shaker (VWR, Radnor, PA) for 72 hours, 37C at approximately

150 rpm and an orbital diameter of 15 mm. After 72 hours of growth, the biofilms had an average

cell density of approximately 2.67 x 106 CFU/cm2. The general setup for shaker biofilms can be

seen in Figure 2. There was more variation in biofilm growth for the shaker biofilms compared to

the drip flow reactor biofilms. Sampling techniques for controls needed to be considered for these

trials. One slide from each dish was reserved for heat shocking at control conditions of 37C. The

heat shock temperatures considered were 50C and 80C, covering the extremes of the

investigative temperature range. The heat shock durations were 2, 10, or 30 minutes. All

quantification was done using direct enumeration.

12
Figure 2 - General setup for shaker biofilm growth. Shaker biofilms are grown using glucose-enhanced
media in an incubator at 37C on an orbital shaker for 72 hours (A). They colonize on frosted glass
microscope slides located in each well of the dish (B). This results in an average bacteria concentration of
2.66 x 106 CFU/cm2.

Drip flow reactor (DFR) biofilm cultures

Biofilms were grown in an incubated (37C) drip flow reactor (DFR), which has ASTM standard

E2647-08 associated with it (BioSurface Technologies Corporation, Bozeman, MT). The DFR

gravitationally allows fresh TSB media to flow through the four reactor wells due to its 10

downward slope. The reactor wells had frosted glass microscope slides in them for biofilm

attachment and growth.

The biofilm formation and growth progressed through two stages, batch and continuous mode.

Batch mode had the incubated DFR at zero incline for 4 hours with a 1:15 mL ratio of inoculum

to fresh TSB in each well to allow bacteria concentration to attach and accumulate on the

microscope slide surface. After batch mode, the DFR was put into continuous mode for 24 hours,

which utilized the 10 incline and peristaltic pump to drip clean TSB through the wells at a rate of

5L/day (Figure 3). This resulted in a consistent biofilm, approximately 50 100 m thick (see

13
Biofilm Characterization via CLSM in results for details) and a cell density of 1.65 x 109

CFU/cm2.

Figure 3 - General setup for drip flow reactor (DFR) biofilms. DFR biofilms were grown in a 4-well
reactor (A) incubated at 37C. TSB media flowed at a rate of 5L/day for 24 hours via a peristaltic pump
through the wells (B). This setup results in an average bacteria concentration of 1.65 x 109 CFU/cm2.

Heat shock system

In order to have instantaneous, homogeneous, thermal shock, the biofilm thermal deactivation

occurred in a water bath heat shock system (Figure 4).

Figure 4 - General setup for heat shock system. Biofilms were transferred to heat shock system via frosted
microscope slides. The system was already stabilized at the desired temperature, then sealed, and
submerged. The well temperature was monitored using thermocouples.

14
The water bath was allowed to stabilize at a given investigative temperature with polystyrene 4-

well rectangular dishes, containing 5 mL of media per well, sealed with parafilm to avoid

contamination and submerged in the bath. The biofilms were transferred to the heat shock system

from the DFR or shaker plates via the frosted microscope slide, on which they had been growing,

into the 4-well heat shock dish. The heat shock dish was resealed, submerged, and maintained at

the elevated temperature for a given time interval. Temperature in the wells was verified and

monitored using thermocouples (two in each of the 4-well dish; type T). Immediately following

the thermal shock, the biofilms were removed via the frosted microscope slide from the 4-well

heat shock dish and placed into a fresh polystyrene 4-well dish with 5 mL of media at room

temperature and then immediately quantified. The heat shock temperatures investigated were

37C (for control evaluation purposes), 50C, 60C, 70C, or 80C and the heat shock durations

were 1, 2, 5, 10, 15, 20, or 30 minutes.

Quantification via direct enumeration

Direct enumeration was used to quantify biofilms because of its ability to precisely quantify

viable bacteria concentrations on a logarithmic scale. Other techniques such as microscopy only

work on an arithmetic scale, limiting quantification reduction to one order of magnitude. The 4-

well dish containing the deactivated biofilms at room temperature were sealed and sonicated

(VWR, Radnor, PA) for 10 minutes, 45 kHz, 240 W in order to disrupt the polysaccharide matrix

and homogenize the bacteria. After sonication, a 100 L sample was taken directly from each of

the 4 wells, serially diluted, and plated on agar-filled petri dishes. Plating was done using the

spread plate technique with glass beads. The petri dishes were incubated at 37C for 24 hours,

and then the CFU were counted for each plated dilution. From the collected plate counts, the

number of viable bacteria in the biofilm were back-calculated based on CFU count per plate and

the dilution factor.

15
Since many dilutions were plated, a counting protocol was set to choose plates on the count range

from 5 to 150 CFUs/plate. If more than one dilution fell in the countable range, the higher plate

count (i.e. lower dilution) was chosen. When calculating the log(CFU/slide), the following

equation was used:

10 5
log(/2 ) = (( 0.1
) () (18.75 2 )) Equation 2

All analyses are performed on the log(CFU) and not the CFU directly since parameters vary on a

logarithmic scale.

Statistical analysis

All statistical analysis was completed using JMP 11.2 statistical software. Means comparison

were done using a one-way ANOVA Tukey-Kramer method with = 0.05. A one-way ANOVA

tests the null hypothesis that means from different groups are the same. The Tukey-Kramer

method compares all pairs of means to test if they are significantly different based on a critical

value of The data is reported as the average log(CFU/cm2) standard deviation.

Results

Growth study

Observing inoculum growth over a 36-hour period showed the inoculum entered and continued in

stationary phase after a 24-hour incubation (37C, 5% CO2). It was consistently at 1.7 x 109

CFU/mL (1.04 x 108 standard deviation), Figure 5. Completing UV-VIS scans over the range of

200 to 800 nm and focusing on OD at 600 nm resulted in OD600 equal to approximately 0.6 after

24-hour incubation.

16
Figure 5 - Relationship between inoculum growth time (h) and CFU/mL. After 24 hours of growth the
inoculum was reproducibly at a concentration of 1.7 x 10 9 CFU/mL (1.04 x 108 standard deviation). n=3.

The inoculum was always cultured for 24-hours, ensuring it had reached a constant stationary

phase concentration of approximately 109 CFU/mL. Having an invariable starting inoculum leads

to more uniform biofilm growth for experimental heat shocks.

Shaker biofilms

P. aeruginosa biofilms were grown in sealed, batch conditions using 4-well polystyrene dishes.

Each well had a frosted microscope slide in it, enabling easy biofilm transfer from the growth

environment to the heat shock system. The plates were agitated using an orbital shaker to

encourage biofilm formation during growth. One slide from each dish was reserved for heat

shocking at control conditions of 37C. The heat shock temperatures considered were 50C and

80C, covering the extremes of the investigative temperature range. The heat shock durations

were 2, 10, or 30 minutes. All quantification was done using direct enumeration.

17
Figure 6 - Control shaker biofilm results. All biofilms were grown using batch mode, 4-well dishes with
glucose-enhanced media for 72 hours, 37C and subjected to thermal protocols at 37C for 2, 10, or 30
min, then quantified using direct enumeration. The blue line error bars represent the standard deviation.
The means were compared using the one-way ANOVA Tukey-Kramer method (=0.05) and showed no
statistical difference between time points. The overall grand mean log(CFU/cm2) for the controls was 6.24
log(CFU/cm2). Minimum quantifiable log reduction threshold is labeled at 1.12.

The controls had a grand mean of 6.24 log(CFU/cm2), i.e. 106.24 or 1,737,800 CFU/cm2, and were

shown to be statically the same by comparing the one-way ANOVA Tukey-Kramer method with

= 0.05 (Figure 6). The biofilms had an average thickness of approximately 25 to 45 m

(measured by CLSM imaging, see Biofilm characterization via CLSM in the Results section

below). Increasing temperature in the heat shock system to 50C or 80C resulted in complete

deactivation of the biofilms. It should be recalled that the counting protocol requires a CFU count

of at least five to be within the minimum threshold limit (see Quantification via direct

enumeration in the Materials & methods section above). Therefore, the minimum quantifiable

log(CFU/cm2) is 1.12. However, the undiluted, heat-shocked biofilms samples were yielding less

than five CFU per plate (often zero CFU) indicating that thermal deactivation reduced the

log(CFU/cm2) lower than the minimum of 1.12. A more robust and denser starting population

was needed in order to be within quantifiable threshold limits. Therefore, biofilms with denser

bacteria loading were pursued using the DFR.

18
Drip flow reactor control biofilms

P. aeruginosa biofilms grown in DFRs were denser and more robust. Their growth conditions

were at 37C using TSB as the media source and a flow rate of 5L/day for 24 hours. Each reactor

had 4-wells with frosted microscope slides in the bottom for biofilm attachment and transferring.

Control biofilms were evaluated in order to verify consistency of processing steps as well as the

starting bacteria concentrations of the heat shock experiments. Post growth, the controls were

transferred to the heat shock system which was stabilized at 37C for a duration of 1, 2, 5, 10, 15,

20, or 30 minutes and then quantified by direct enumeration. Comparing the means using the one-

way ANOVA Tukey-Kramer method ( = 0.05) at each time point showed there was no statistical

difference between controls (Figure 7). The grand mean for the controls was 8.553 log(CFU/cm2).

The average thickness of the biofilms varied from 50 100 m (measured by CLSM imaging, see

Biofilm characterization via CLSM in the Results section below).

Figure 7 - Control results for DFR biofilms. All biofilms were grown using a DFR with TSB for 24 hours,
37C and subjected to thermal protocols at 37C for 1, 2, 5, 10, 15, 20, or 30 min, then quantified using
direct enumeration. The blue line error bars represent the standard deviation. The means were compared
using the one-way ANOVA Tukey-Kramer method (=0.05) and showed no statistical difference between
time points. The overall grand mean log(CFU/cm2) for the controls was 8.553. Minimum quantifiable log
reduction threshold is labeled at 1.12.

19
The DFR biofilms produced were consistent and 350 times denser, based on bacteria

concentration, than the shaker biofilms and more than twice as thick. These biofilms were also

visually different than shaker biofilms. They had a tackier matrix and covered the entire

microscope slide, whereas the shaker biofilms were easily sheared and patchy over the

microscope slide area.

Biofilm characterization via CLSM

Using CLSM to characterize biofilms showed there was a difference in thickness and architecture

between shaker and DFR biofilms. Shaker biofilms were approximately 25 to 45 m thick with a

control grand mean bacteria concentration of approximately of 6.24 log(CFU/cm2), while DFR

biofilms were approximately 50 to 100 m thick with a control grand mean 8.59 log(CFU/cm2).

The shaker biofilms were also less viscous and only covered patchy sections of the microscope

slide sample, while the DFR biofilms produced higher coverage by colonizing the entire slide.

Figure 8A and 8B depict examples of controls for a shaker and DFR biofilm, respectively,

representing the difference in coverage and thickness.

20
Figure 8 - CLSM control biofilms. Live bacteria fluoresce green and dead bacteria fluoresce red. Shaker
biofilms (A) were approximately 25 to 45 m thick with patchy coverage over the microscope slide, while
DFR biofilms (B) were 50 to 100 m thick with dense coverage over the entire slide.

To show the contrast between heat shocked and control samples, DFR biofilms were heat

shocked at 80C for 30 minutes and imaged as described above. Figure 9A shows the control that

was heat shocked at 37C for 30 minutes, while Figure 9B displays the heat shocked biofilm at

80C for 30 minutes. The level of deactivation at this extreme time and temperature combination

is obviously high, as seen by the amount of red (dead) fluorescing bacteria.

21
Figure 9 Representative confocal micrographs of a DFR control biofilm at 37C for 30 minutes (A)
compared to a heat shocked DFR biofilm at 80C and 30 minutes (B). Live bacteria fluoresce green and
dead bacteria fluoresce red. This image displays the high level of deactivation from the extreme heat shock
time and temperature combination.

Shaker biofilms were also heat shocked for various durations at 80C in order to verify the visual

decrease in live bacteria as the heat shock protocol was increased. Figure 10A, 10B, and 10C

displays 80C heat shocked shaker biofilms for 1, 15, and 30 minute times respectively. As the

heat shock time is increased, the amount of red fluorescing bacteria is increased and the amount

of green fluorescing bacteria is decreased. This further supports that thermal shock of biofilms is

an appropriate means of deactivation, and that specific time and temperature combinations result

in different levels of deactivation.

22
Figure 10 Confocal micrographs of shaker biofilms heat shocked at 80C for 1, 15, and 30 minutes. Live
bacteria fluoresce green and dead bacteria fluoresce red. As the heat duration increases from 1 minute (A)
to 15 minutes (B) to 30 minutes (C), the amount of red fluorescing bacteria increases and the amount of
green fluorescing bacteria decreases.

DFR Heat shock biofilms

Heat shock biofilms experienced the same growth and thermal protocols as the control biofilms,

but differed by elevating the temperature to 50C, 60C, 70C, or 80C. At each temperature,

durations of 1, 2, 5, 10, 15, 20, or 30 minutes were evaluated (Table 1).

Table 1 - Average log(CFU/cm2) standard deviation of DFR biofilms as a function of heat shock
temperature and duration. Heat shocks at 37C were comparable controls. In general as heat shock
temperature or time increases, the average log(CFU/cm2) decreases.

Since complete deactivation of the DFR biofilms was never achieved, overall trends of log

reduction and the sensitivity to heat shock time and temperature combinations was quantifiable

via direct enumeration (Figure 11). In general, as heat shock time or temperature increased, the

average log(CFU/cm2) decreased.

23
 Time (min)

Figure 11 Average log(CFU/cm2) for each DFR biofilm heat shock time and temperature combination. In
general, as heat shock temperature or time is increased, the average log(CFU/cm2) decreases. Minimum
threshold log reduction labeled at 1.12

Discussion

In order to establish thermal deactivation as an innovative treatment for biofilm infected medical

devices, it was necessary to determine the time and temperature combinations needed for death to

occur and the log reduction trends that develop from them. Beginning with the shaker biofilms

was an encouraging start because it was confirmed that heat alone could be an appropriate means

of biofilm eradication.

There were issues, however, that needed to be addressed beyond the shaker biofilm trials. The

shaker biofilms, once heat shocked, resulted in large rates of log reduction of viable bacteria. The

thermal protocols were so successful at deactivating the biofilm regardless of the time or

temperature of the heat shock that it resulted in log(CFU/cm2) below the minimum quantifiable

threshold of 1.12. There was not enough quantification sensitivity to establish what levels of log

24
reduction heat shocks were actually accomplishing. Another issue with the shaker biofilms was

the high level of enumeration variability post heat shock, resulting in large standard deviations of

log(CFU/cm2). This could be attributed to variability in the biofilms matrix concentration.

Visually the shaker biofilms differed in levels of clarity and viscosity based on changes in the

amount of matrix grown. Many of a biofilms resistance factors can be associated with the matrix

transport limitation, physiological gradients, QS signaling, etc. Having less matrix, could lead

to the cells behaving more as the planktonic phenotype as opposed to the biofilm phenotype. The

bacteria would not be triggering the stress response or up-regulating efflux pumps. Thermal shock

would easily be able to sterilize the colony. As matrix levels increased, more resistance would

develop resulting in the large standard deviations seen. Switching to the more standardized

DFR growth environment addressed both the quantification sensitivity and varying matrix issue.

DFR biofilms were denser (350 times higher grand mean for control bacteria concentration) than

shaker biofilms and visually more viscous and tackier (more than twice as thick). The DFR

controls verified the starting bacteria density to be statistically the same (Figure 7). Adding in

thermal heat shock further verified that the biofilms were more robust than the shaker biofilms

because the quantification sensitivity increased. Complete thermal deactivation of the biofilm was

not seen with DFR biofilms because the starting concentration of matrix and bacteria were higher.

Ranging the temperature from 50C to 80C and heat shock durations from 1 to 30 minutes

resulted in general trends showing log reduction in viable bacteria (Figure 11). The highest

deactivation was approximately 6.6 average log reduction, occurring at the high end of the time

and temperature combinations of 80C for 30 minutes. The lowest deactivation was

approximately 0.1 average log reduction, occurring at the low end of the time and temperature

combination of 50C and 1 minute. This was expected, and supports the hypothesis that biofilms

are susceptible to thermal shock. Now when choosing a time and temperature protocol, one could

25
predict the approximate amount of log reduction that will be seen in a similar P. aeruginosa

biofilm.

Increasing either heat shock duration or temperature was successful at decreasing log(CFU/cm2),

however, temperature had a much more dramatic difference than increasing time alone.

Comparing the means using a one-way ANOVA Tukey-Kramer method showed that changing

the heat shock time was not statistically different or significant for log(CFU/cm2) (Figure 12A),

while doing the same means comparison of the temperature increase shows a statistical difference

(Figure 12B). This concludes that log reduction is more susceptible to temperature changes. As

this treatment method is optimized, the temperature effect will be taken into consideration. The

high temperature, short duration heat shocks may be more appropriate for patients, than the lower

temperature and longer duration heat shocks.

26
Figure 12 - Effect of heat shock duration (A) and temperature (B) on log(CFU/cm2). Heat shock time
duration and temperature are both important when reducing log(CFU/cm2). One-way ANOVA mean
comparison using the Tukey-Kramer method (alpha = 0.05) shows there is a larger significant difference
with regards to temperature than time.

When linear expressions are correlated to log(CFU/cm2) vs. temperature at each heat shock

duration, a strong dependency on temperature can again be observed (Figure 13). The linear

regressions have R2 values ranging from 0.71 to 0.9 and show similar slopes as temperature is

increased.

27
Figure 13 - Linear correlations of log(CFU/cm2) vs. temperature for each heat shock duration. As time is
increased, slopes remain similar between heat shock durations.

Further data analysis shows that plotting log(CFU/cm2) vs. log(time) results in better linear

regression than just plotting against time. An example for 60C is found in Figure 14. The

log(CFU/cm2) vs. time tracks linearly with an R2 value of 0.49, while log(CFU/cm2) vs. log(time)

has an R2 value of 0.63.

28
Figure 14 - Representative linear regressions for log(CFU/cm2) vs. time (A) and log(time) (B) for
60C. Better R2 value of 0.63 is seen for the plot vs. log(time) as compared to time with an R2 of
0.49.

29
When doing a similar analysis as Figure 13 while constraining the intercept to the grand mean

control value of 8.553, results in strong linear relationships with R2 values from 0.72 to 0.96.

These slopes were then extracted and plotted against log(time) resulting in a linear relationship

with R2 of 0.98 (Figure 15). The 15 minute heat shock duration point was excluded as an outlier

because of possible effects of a heat shock response.

Figure 15 - Correlation of cell death temperature dependence to log(time). 15 minute heat shock
duration point was excluded from the trendline shown as an outlier due to a possible heat shock
response.

These relationships indicate that the experimental results can be expressed by:

log(2 ) = log(2 )0 [0.079 + 0.046 log()]( 37) (Equation 3)

where T is the thermal shock temperature in C and t is the exposure time in minutes. Equation 3

may also be expressed:

(2 ) = (2 )0 100.079(37) 0.046(37) (Equation 4)

which more clearly shows a traditional Arrhenius dependence on temperature. Equations 3 and 4

show that temperature will have a greater effect than time on the CFU/cm2 as each is increased.

30
The amount of log reduction in viable bacteria count from thermal deactivation is as high, if not

higher, than what has been seen in developing biofilm treatment methods currently underway.

Many of the other treatment methods also depend on complementary means of treatment to have

applicable effects on viable cell counts. For example, targeting an electric current to a biofilm

alone resulted in no log reduction of live bacteria, but supplementing antibiotic with electrical

current had a 0.5 log reduction (Jass and Lappin-Scott 1996). Ultrasound only had merely a 1 log

reduction on biofilm viability (P. aeruginosa specifically) (Ensing, Neut et al. 2006). Other

harsher biofilm treatments, achieve slightly higher deactivation, but are difficult to use in a

medial implant application. For example, chlorine is a common biofilm treatment in the water

treatment and food industries having a 3 log reduction for Salmonella spp. when exposed to 100

ppm Cl2 for 5 min (Joseph, Otta et al. 2001). Oral plaque biofilms have been reduced 2 log using

photodynamic therapy (Wood, Metcalf et al. 2006). All of these treatments, however, work in

tandem with other treatments, are more severe, and do not reach the same level of log reduction

as shown using heat alone.

Establishing the time and temperature combinations needed for various levels of viable bacteria

log reduction was the first step towards the long-term goal of developing an innovative biofilm

treatment method. Being able to characterize and predict reduction trends is important. It should

be questioned, however, if these particular robust DFR P. aeruginosa biofilms are representative

of what is seen of biofilms in vivo. Biofilms are known for being a challenge to quantify their

bacterial load on implants. Current techniques include various microscopy techniques: bright-

field, epifluorescence, scanning electron, confocal laser scanning (Sawhney and Berry 2009), but

these are not able to be done in vivo. The shaker biofilms could be more representative of in vivo

biofilm thickness, bacteria concentration, and architecture. This would result in a less extreme

heat shock protocol needed to eradicate the biofilm infection to the same or greater log reduction.

31
Either way, thermal deactivation of biofilms appears to be a more encouraging method than what

is currently available.

Going forward with this treatment will lead to addressing synergistic effects that would be seen

on log reduction when bringing in another method of deactivation. This heat shock approach is

unique because it is one of the only mitigation techniques being explored that does not employ a

complementary approach. Adding in, for example, antibiotics might shift the time and

temperature combinations needed to reach the same levels of log reduction. A final issue to

address would be the effect of multiple pathogens on the thermal protocol. It is often seen that

biofilms are composed of more than one species of bacteria and each of these have a different

mechanism and reaction to treatment methods. Considering antibiotics and multiple strains would

also mimic clinical practices better since one would rarely experience a single strain biofilm or

develop an infection and not be given antibiotics as part of treatment. Thermal deactivation is

known to be effective for all planktonic strains and expectantly this would also be true for biofilm

strains.

Conclusions and future investigations

Biofilm infection of implanted medical devices is an ongoing problem with no easy or readily

available solution. While treatment methods have been pursued, none have been able to fully

eradicate the issue. Complicating the problem is the fact that biofilms continue to increase in

resistance to typical treatments such as antibiotics. Thermal deactivation would be an innovative

method to mitigate biofilm infections. It uses heat to kill biofilms at the device interface. This

heat would eventually be supplied wirelessly by immobilizing magnetic nanoparticles in a device

coating and exposing an alternating magnetic field outside the body of the patient.

32
It was shown that varying heat shocks from 50C to 80C and durations of 1 to 30 minutes could

deactivate DFR grown P. aeruginosa biofilms. In particular this research had the following

results that align with the objectives discussed in the Long-term project goal section:

1. Biofilms were cultured in two different ways, shaker and DFR. They were both produced

from an inoculum on the order of 109 CFU/mL that was established through a growth

study.

2. Shaker biofilms resulted in an architecture that was less viscous and had less microscope

slide coverage than DFR biofilms. Shaker biofilms were 25 to 45 m thick and had an

overall control concentration of 6.24 log(CFU/cm2) while DFR biofilms were 50 to 100

m thick and an overall control concentration of 8.553 log(CFU/cm2). Thickness was

evaluated using CLSM and viable bacteria were quantified using direct enumeration.

3. A water bath system was developed to heat shock shaker and DFR biofilms while

minimizing thermal inertia and instantaneously applying a thermal shock. Shaker

biofilms were heat shocked at the control temperature of 37C and the extremes of the

investigated temperature range of 50C and 80C and heat durations of 2, 10, or 30

minutes. DFR biofilms were heat shocked at the control temperature of 37C and 50C,

60C, 70C, and 80C and times of 1, 2, 5, 10, 15, 20, and 30 minutes.

4. Heat shocking shaker biofilms resulted in a log reduction than was not quantifiable by

direct enumeration (log(CFU/cm2) 1.12; i.e. plate count less than 5 CFUs). DFR

biofilms were therefore perused in order to use biofilms with higher initial bacteria

concentration for a greater possibility of log reduction quantification. Heat shocked DFR

biofilms had log reductions ranging from 0.1 to 6.6 depending on the time and

temperature combination used in the thermal protocol. Further data analysis showed that

a linear regression could be fit for cell death temperature dependence vs. log(time),

resulting in Equations 3 or 4. This shows a strong temperature dependence on log

33
 reduction and gives the ability to approximate deactivation for the DFR biofilms, given a

time and temperature combination.

Future investigations will center on optimizing this treatment method. Things to be considered

include what is representative of an in vivo biofilm, what synergistic effects antibiotics would

contribute, and what the addition of different bacteria strains would have on the thermal protocol.

This research addresses a large problem in the medical world and leads to the long-term goal of

developing an innovative treatment for biofilm infected medical devices.

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