Honours Thesis 2015 Development of in vitro infection ... · Figure 1.2 Schematic diagram of K....

Honours Thesis 2015

Development of in vitro infection model

methods for Gram-negative bacteria to

assess potential broad-spectrum anti-

virulence inhibitors

Naomi Jebakumar

Bachelor of Science in Biomedical Science

School of Veterinary and Life Sciences

Murdoch University

Declaration

I declare this thesis is my own account of my research and contains as its main content

work which has not been previously submitted for a degree at any tertiary education

institution.

Naomi Jebakumar

iii

Abstract

Bacteria exhibit common virulence targets which can be inhibited for potential

broad-spectrum activity across a range of bacterial infections. The macrophage

infectivity potentiator (Mip) is a bacterial protein that has peptidylprolyl cis/trans

isomerase activity and is part of the FK506-binding protein subgroup, in the superfamily

of immunophilins. They have previously been identified to be involved in intracellular

virulence of Legionella pneumophila and Burkholderia pseudomallei and inhibition of the

protein with pipecolic acid derived small-molecule inhibitors resulted in a decrease in

virulence. Due to the highly conserved nature of Mip proteins it is believed that these

proteins exist in Klebsiella pneumoniae and Burkholderia cenocepacia; two pathogens

contributing to a rise in multi-drug resistant infections. Therefore, this study aims to

examine the potential broad-spectrum activity of the Mip inhibitors in K. pneumoniae

and B. cenocepacia by developing in vitro model methods for inhibitor evaluation.

The presence of putative Mip proteins in K. pneumoniae and B. cenocepacia were

confirmed with the use of online bioinformatics tools. The Mip inhibitors were then

examined by first developing in vitro cell based methods to identify the magnitude at

which K. pneumoniae and B. cenocepacia adhered to macrophages, and internalised,

survived and replicated within the macrophages. An optimised adherence assay, and

internalisation, survival and replication assay were then used to test the efficacy of the

Mip inhibitors in K. pneumoniae and B. cenocepacia.

The results obtained demonstrated that K. pneumoniae and B. cenocepacia were

able to adhere to the macrophages, however, the concentration at which they adhered

varied between strains. Only two strains of K. pneumoniae were internalised into the

macrophages and only one strain was able to replicate at low concentrations within the

iv

macrophages during the 24 hours post-infection. All strains of B. cenocepacia were

internalised into the macrophages, however, only one strain suggested replication

within the macrophages during the 24 hours post-infection. The Mip inhibitors had little

effect on adherence of K. pneumoniae to macrophages, however, trends in the data

suggested that the Mip inhibitors had an effect on the intracellular virulence of K.

pneumoniae and B. cenocepacia. Therefore from this preliminary study, it seems that

Mip inhibitors have potential broad-spectrum activity in intracellular virulence of these

pathogens. These findings provide insight into Mip proteins in K. pneumoniae and B.

cenocepacia and the potential they have as common bacterial drug targets.

v

Acknowledgements

Firstly, I would like to thank my supervisors Dr. Tim Inglis, Dr. Mitali Sarkar-Tyson

and Dr. Wayne Greene for their continuous support and input over the course of this

year. I have learned so much and will always be grateful for the opportunity and

experiences I have received. A special thank you to Dr. M. Sarkar-Tyson for her

encouragement and motivation throughout this year. Thank you for always being there

when I needed a question answered and for pushing me to reach further. I am so grateful

to have a supervisor who went out of her way to make this year an enjoyable one!

Thank you to Professor Ulrike Holzgrabe and her team for allowing me to use the

Mip inhibitors as part of my study; to my lab group and particularly Jarrad, for teaching

me the ropes of research and for constantly putting up with all my requests; to Shaxx

for his help throughout the year, especially with statistics; to Nicole for your friendship,

encouragement and help, and for routinely organising lab lunches! Thank you to my

honours buddies, Courtney and Katherine, for all the laughs, rants and “secret honours

meetings.” This year would not have been half as fun without you and I am so grateful

for the friendships we have made, and for the constant encouragement you have both

provided me.

Thank you to my amazing friends for listening to me talk about science and for

letting me vent when I needed to; to my loving family for their ongoing support,

especially my parents who have always pushed me to be the best I can be. Above all,

thank you Lord Jesus, for being there during the highs and lows of this year. Your grace

has brought me through.

vi

Table of Contents

Abstract ........................................................................................................................................ iii

Acknowledgements ....................................................................................................................... v

Table of Contents ......................................................................................................................... vi

List of Figures ................................................................................................................................. x

List of Tables ................................................................................................................................ xii

Abbreviations ............................................................................................................................. xiii

1. Introduction .............................................................................................................................. 2

1.1 The genus Klebsiella ............................................................................................................ 2

1.1.1 Klebsiella pneumoniae ................................................................................................. 2

1.1.1.1 Disease and clinical presentation.......................................................................... 4

1.1.1.2 Treatment and vaccine development ................................................................... 5

1.1.1.2.1 Antibiotic resistance ...................................................................................... 6

1.1.1.3 Epidemiology ......................................................................................................... 7

1.1.1.4 Virulence factors ................................................................................................... 9

1.1.1.4.1 Capsular polysaccharides ............................................................................... 9

1.1.1.4.2 Pili ................................................................................................................... 9

1.1.1.4.3 Siderophores ................................................................................................ 10

1.1.1.4.4 Serum resistance and lipopolysaccharides .................................................. 11

1.2 The genus Burkholderia .................................................................................................... 13

1.2.1 The Burkholderia cepacia complex ............................................................................ 13

1.2.1.1 Disease and clinical presentation........................................................................ 15

1.2.1.2 Treatment ........................................................................................................... 16

1.2.1.2.1 Antibiotic resistance .................................................................................... 17

1.2.1.3 Epidemiology ....................................................................................................... 20

1.2.1.4 Virulence factors of Burkholderia cenocepacia .................................................. 22

1.2.1.4.1 Quorum sensing ........................................................................................... 22

1.2.1.4.2 Siderophores ................................................................................................ 22

1.2.1.4.3 Motility and adherence ................................................................................ 23

vii

1.2.1.4.4 Polysaccharides ............................................................................................ 24

1.3 Macrophage infectivity potentiator proteins ................................................................... 25

1.4 Virulence targets for novel inhibitors ............................................................................... 27

1.4.1 Quorum sensing inhibitors ......................................................................................... 27

1.4.2 Cell division inhibitors ................................................................................................ 28

1.4.3 Mip inhibitors ............................................................................................................. 29

1.5 In vitro models for novel inhibitor testing ........................................................................ 30

1.5.1 Biofilm models ........................................................................................................... 31

1.5.2 3D tissue-engineering models.................................................................................... 31

1.5.3 Cell based models ...................................................................................................... 32

1.6 Project aim ........................................................................................................................ 33

1.7. Significance ...................................................................................................................... 34

2. Materials and methods .......................................................................................................... 36

2.1 Materials ........................................................................................................................... 36

2.1.1 Bacterial strains and mammalian cell line ................................................................. 36

2.1.2 Growth media and supplements ............................................................................... 37

2.1.3 Antibiotics and chemicals .......................................................................................... 37

2.1.4 Bioinformatic tools and software .............................................................................. 39

2.2. Methods ........................................................................................................................... 39

2.2.1 Bacterial recovery from glycerol stock and maintenance ......................................... 39

2.2.2 Mammalian tissue culture ......................................................................................... 39

2.2.2.1 Cell revival ........................................................................................................... 39

2.2.2.2 Cell passage and maintenance ............................................................................ 40

2.2.3 Cell infection assays ................................................................................................... 40

2.2.3.1 Cell preparation for in vitro assays ..................................................................... 40

2.2.3.2 Bacterial growth for in vitro assays ..................................................................... 40

2.2.3.4 Dilution of overnight bacterial culture for enumeration and MOI calculations . 41

2.2.3.4.1 Overnight bacterial culture dilutions for in vitro assays .............................. 41

2.2.3.4 In vitro assays ...................................................................................................... 42

viii

2.2.3.4.1 Adherence assays ......................................................................................... 42

2.2.3.4.2 Internalisation, survival and replication assays ........................................... 43

2.2.3.4.3 Mip inhibitor testing in adherence assays, and internalisation, survival and

replication assays ........................................................................................................ 44

3. Bioinformatic confirmation of putative Mip homologues in K. pneumoniae and B.

cenocepacia, and quantitative determination of MOIs ............................................................ 46

3.1 Strains ............................................................................................................................... 46

3.2 Identification of Mip homologues in K. pneumoniae and B. cenocepacia through

bioinformatic analysis ............................................................................................................. 46

3.3 Overnight dilution experiments ........................................................................................ 48

3.3.1 K. pneumoniae dilution .............................................................................................. 48

3.3.2 B. cenocepacia dilution .............................................................................................. 50

4. The assessment of K. pneumoniae and B. cenocepacia adherence to RAW264.7

macrophage cells as a model for inhibitor evaluation .............................................................. 53

4.1 K. pneumoniae adherence to RAW264.7 macrophage cells ............................................. 54

4.2 B. cenocepacia adherence to RAW264.7 macrophage cells ............................................. 56

5. The assessment of K. pneumoniae and B. cenocepacia internalisation, survival and

replication within RAW264.7 macrophage cells as a model for inhibitor evaluation ............. 59

5.1 K. pneumoniae internalisation, survival and replication within RAW264.7 macrophage

cells ......................................................................................................................................... 60

5.2 B. cenocepacia internalisation, survival and replication within RAW264.7 macrophage

cells ......................................................................................................................................... 62

6. The effect of Mip inhibitors on adherence and internalisation, survival and replication of

K. pneumoniae and B. cenocepacia in RAW264.7 macrophage cells ....................................... 65

6.1. K. pneumoniae adherence to RAW264.7 macrophage cells with the Mip inhibitors ...... 67

6.2 B. cenocepacia adherence to RAW264.7 macrophage cells with the Mip inhibitors ....... 69

6.3 K. pneumoniae internalisation, survival and replication within RAW264.7 macrophage

cells with the Mip inhibitors ................................................................................................... 69

6.4 B. cenocepacia internalisation, survival and replication within RAW264.7 macrophage

cells with the Mip inhibitors ................................................................................................... 73

7. Discussion ............................................................................................................................... 76

ix

7.1 Bioinformatic confirmation of putative Mip homologues in K. pneumonia and B.

cenocepacia ............................................................................................................................ 78

7.2 The assessment of K. pneumoniae and B. cenocepacia adherence to RAW264.7

macrophage cells as a model for inhibitor evaluation ............................................................ 79

7.2.1 K. pneumoniae adherence to RAW264.7 macrophage cells ...................................... 80

7.2.2 B. cenocepacia adherence to RAW264.7 macrophage cells .......................................... 82

7.3 The assessment of K. pneumoniae and B. cenocepacia internalisation, survival and

replication within RAW264.7 macrophage cells as a model for inhibitor evaluation ............ 83

7.3.1 K. pneumoniae internalisation, survival and replication within RAW264.7

macrophage cells ................................................................................................................ 84

7.3.2 B. cenocepacia internalisation, survival and replication within RAW264.7

macrophage cells ................................................................................................................ 86

7.4 The assessment of the effects of Mip inhibitors on adherence, and internalisation,

survival and replication of K. pneumoniae and B. cenocepacia within RAW264.7 macrophage

cells ......................................................................................................................................... 87

7.4.1 K. pneumoniae and B. cenocepacia adherence to RAW264.7 macrophage cells with

the Mip inhibitors ............................................................................................................... 88

7.4.2 K. pneumoniae and B. cenocepacia internalisation, survival and replication within

RAW264.7 macrophage cells with the Mip inhibitors ........................................................ 90

7.5 Limitations and future work ............................................................................................. 93

7.6 Conclusion ......................................................................................................................... 94

8. Reference List ......................................................................................................................... 96

Appendix ................................................................................................................................... 113

x

List of Figures

Figure 1.1 Global distribution of K. pneumoniae carbapenemase strains by country

of origin

Figure 1.2 Schematic diagram of K. pneumoniae virulence factors

Figure 1.3 Antibiotic resistance mechanisms of the Burkholderia cepacia complex

Figure 3.1 Alignment of the L. pneumophila, B. pseudomallei, B cenocepacia and K.

pneumoniae Mip sequence

Figure 3.2 Dilution of K. pneumoniae strain MGH78578 overnight cultures

Figure 3.3 Dilution of K. pneumoniae strain ST23.1 overnight cultures

Figure 3.4 Dilution of B. cenocepacia strain 164 overnight cultures


Figure 4.1 Adherence assay model

Figure 4.2 Concentration of K. pneumoniae that adhered to RAW264.7

macrophage cells

Figure 4.3 Concentration of B. cenocepacia that adhered to RAW264.7

macrophage cells

xi

Figure 5.1 Internalisation, survival and replication assay model

Figure 5.2 Concentration of K. pneumoniae that internalised, survived and

replicated within RAW264.7 macrophage cells

Figure 5.3 Concentration of B. cenocepacia that internalised, survived and


Figure 6.1 Mip inhibitor testing models


macrophage cells with the Mip inhibitors

Figure 6.3 Concentration of K. pneumoniae strain ST628 that were internalised

into RAW264.7 macrophage cells with the Mip inhibitors

Figure 6.4 Concentration of K. pneumoniae strain ST14 that internalised, survived

and replicated within RAW264.7 macrophage cells with the Mip

inhibitors

Figure 6.5 Concentration of B. cenocepacia strain 165 that internalised, survived

and replicated within RAW264.7 macrophage cells with the Mip

inhibitors

xii

List of Tables

Table 1.1 Overview of the Burkholderia cepacia complex

Table 1.2 Distribution of the Burkholderia cepacia complex in six cystic fibrosis

population studies

Table 2.1 K. pneumoniae strains used in this study

Table 2.2 B. cenocepacia strains used in this study

Table 2.3 Media and supplements used in this study

Table 2.4 Antibiotics and chemicals used in this study

Table 2.5 Dilutions of overnight bacterial cultures in media

xiii

Abbreviations

% Percent

°C Degrees Celsius

3D Three-dimensional

3x Three times

α Alpha

ATP Adenosine triphosphate

β Beta

Bcc Burkholderia cepacia complex

BLAST Basic local alignment search tool

CFU Colony-forming units

CO2 Carbon dioxide

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

ESBL Extended spectrum β-lactamase

FBS Foetal bovine serum

FKBP FK506-binding proteins

ICU Intensive care unit

kDa Kilodalton

KPC K. pneumoniae carbapenemase

L-15 Leibovitz’s L-15 Medium

LB Luria-Bertani

MDR Multi-drug resistant

xiv

mg Milligram

min Minute(s)

Mip Macrophage infectivity potentiator

mL Millilitre

MOI Multiplicity of infection

NCBI National Centre for Biotechnology Information

nm Nanometre

NMR Nuclear magnetic resonance

PBS Phosphate-buffered saline

PPIase Peptidylprolyl cis/trans isomerase

RND Resistance nodulation division

SEM Standard error of the mean

TAA Trimeric autotransporter adhesin

μg Microgram

UK United Kingdom

μL Microlitre

μm Micrometre

USA United States of America

WHO World Health Organisation

x g Centrifugal force

1

Chapter One

Introduction

2

1. Introduction

1.1 The genus Klebsiella

The genus Klebsiella is part of the Enterobacteriaceae family and was named after

the German microbiologist Edwin Klebs in 1885 to honour his work (Brisse, Grimont &

Grimont 2006). Klebsiella are facultative anaerobic, encapsulated, non-motile, Gram-

negative bacilli that have variable fermentative and biochemical activities based on the

species (Chapman 1946). They are isolated from human respiratory, gastrointestinal and

urinary tracts but are also widely seen in the environment (Chapman 1946). In the genus

Klebsiella, Klebsiella pneumoniae and Klebsiella oxytoca are the most medically

important species causing opportunistic infections in humans (Hidron et al. 2008). K.

pneumoniae is of particular interest due to an increase in multi-drug resistant (MDR)

strains and hospital cases.

1.1.1 Klebsiella pneumoniae

K. pneumoniae was first described in 1882 by Carl Friedländer who found the

bacterium in the lungs of a patient that died of pneumonia (Brisse, Grimont & Grimont

2006). Prior to this, two other species were also described; Klebsiella rhinoscleromatis

and Klebsiella ozaenae. Due to the inability to distinguish between these three species

based on DNA relatedness, they were collectively placed as subspecies under the type

species K. pneumoniae (Brisse, Grimont & Grimont 2006). For the remainder of this

thesis K. pneumoniae will be referring to the species K. pneumoniae subspecies

pneumoniae.

Clinical isolates of K. pneumoniae have an optimum growth temperature of 35 - 37

°C on common laboratory media such as Luria-Bertani (LB) media, blood agar plates and

3

tryptic soy media. K. pneumoniae produce white, glistening, convex and smooth mucoid

colonies on agar plates due to a polysaccharide capsule (Breed, Murray & Smith 1957;

Ristuccia & Cunha 1984). They are rod shaped with rounded ends, measuring 0.3 – 0.5

by 5.0 μm (Breed, Murray & Smith 1957). K. pneumoniae are capable of fermenting

lactose and can utilise citrate as their sole carbon source (Ristuccia & Cunha 1984). They

are also capable of using ammonia as their sole nitrogen source, converting nitrates into

nitrites (Breed, Murray & Smith 1957).

Like all Gram-negative bacteria, K. pneumoniae has a complex bi-layered cell wall

(Wojciechowski 2006). The inner component of the cell wall is made up of polymer

peptidoglycan molecules; a composite of long strands of glycan and crosslinked

stretchable peptides (Huang et al. 2008). The outer component of the cell wall is made

up of lipopolysaccharide molecules and has toxic activity which is responsible for many

symptoms associated with Gram-negative infections (Wojciechowski 2006). K.

pneumoniae also contain pili (fimbriae) on the outer membrane of the cell wall which

assists the bacteria in adhering to cells and aids colonisation in the host (Fader, Avots-

Avotins & Davis 1979).

The most common way to classify K. pneumoniae is by using the capsular K-antigen

serotyping method (Brisse, Grimont & Grimont 2006). The first two capsular serotypes

identified were by Julianelle (1926) who looked at various serological tests such as

agglutination and precipitin reactions in different K. pneumoniae strains. Since his

pioneering ground work, 82 capsules (K1 – K82) have been identified. However in 1977,

5 capsular serotypes were deleted which include K73 and K75 – K78 (Ørskov & Fife-

Asbury 1977). 77 capsular serotypes remain and are part of the international K-typing

scheme (Ørskov & Fife-Asbury 1977).

4

1.1.1.1 Disease and clinical presentation

K. pneumoniae can cause severe and sometimes fatal infections. These include liver

abscesses, pneumonia, urinary tract infections and septicaemia (Ristuccia & Cunha

1984). MDR K. pneumoniae infections have been associated with adverse clinical

outcomes, increased mortality and prolonged hospitalisation (Pons et al. 2015). The

capsular type of K. pneumoniae is related to the severity of infection (Cortés et al. 2002;

Mizuta et al. 1983). K1 and K2 capsular K. pneumoniae are the most commonly isolated

strains from patients (Fang et al. 2007). A new and emerging hypervirulent

(hypermucoviscous) phenotype of K. pneumoniae that was first identified in the mid

1980’s has been reported to cause serious and life-threatening invasive infections (Liu,

Cheng & Lin 1986). A devastating characteristic of this phenotype is the ability to cause

secondary complications by spreading from the site of infection to distant parts of the

body via unknown mechanisms (Liu, Cheng & Lin 1986).

Clinical presentations associated with K. pneumoniae infections in pneumonic

patients include the sudden appearance of cough, fever, rigors and pleuritic chest pain,

shallow respiration, as well as thick, bloody sputum due to the necrotising inflammatory

process caused by the bacteria (Prince et al. 1997; Sheff 2000). Urinary tract infections

cause frequent urination, burning sensation and blood or pus in the urine (Sheff 2000).

Recurrent urinary tract infections can damage renal function and even after the first

episode of infection, renal scarring is seen in up to 57% of cases (Lin et al. 2014).

Pyogenic liver abscesses present non-specific symptoms such as fever, fatigue, nausea

and anorexia (Casella et al. 2009; Pope et al. 2011). Hypervirulent K. pneumoniae strains

are capable of causing severe invasive liver abscesses with extrahepatic complications,

5

including central nervous system involvement, necrotising fasciitis, meningitis and

endophthalmitis (Siu et al. 2012).

Infections caused by K. pneumoniae are seen in both community-acquired settings

and nosocomial settings, and are typically observed in patients who are

immunocompromised. Risk factors associated with community-acquired infection

include alcoholism, diabetes, biliary disease, cancer, acute renal failure and heart

disorders (Jong et al. 1995; Thomsen, Jepsen & Sørensen 2007; Falagas et al. 2007). Risk

factors associated with nosocomial K. pneumoniae infections include invasive surgery,

burn wounds, catheters, mechanical ventilation, antimicrobial therapy, extremes of age

and duration of stay at the hospital (Ulu et al. 2015; Brisse, Grimont & Grimont 2006).

Vardakas et al. (2015) found that mortality rates increased with prolonged stay in the

intensive care unit (ICU). Paediatric wards are of particular risk especially premature

infants in ICU’s (Podschun & Ullmann 1998).

1.1.1.2 Treatment and vaccine development

Treatment for K. pneumoniae infections are based on whether the strain exhibits

antibiotic resistance or not. Strains which are not MDR can be treated with

aminoglycosides and quinolones (Wojciechowski 2006). Extended spectrum β-

lactamase (ESBL) K. pneumoniae strains can be treated with carbapenem antibiotics.

However, in the case of K. pneumoniae carbapenemase (KPC) strains, combination

therapy of colistin with either tigecycline or imipenem may be needed for possible

synergistic effects as last resort drug treatments (Ah, Kim & Lee 2014; Katsiari et al.

2015; Munoz-Price et al. 2013).

6

Vaccine efforts have continued over the past decade. The first antibody

preparations were against the capsular polysaccharide using inactivated whole cells,

ribosomal preparations and cell surface preparations (Riottot, Fournier & Pillot 1979;

Fournier et al. 1981). Cryz, Fürer & Germanier (1984) found that anti-polysaccharide

antibodies produced against a highly purified antigen provided high levels of protection

from fatal K. pneumoniae burn wound sepsis. A human trial of this vaccine found that it

was safe and immunogenic (Cryz, Fürer & Germanier 1985). Other vaccine efforts

include active immunisation via whole cell vaccines, killed/attenuated vaccines, protein

based vaccines, ribosomal vaccines and conjugate vaccines (Ahmad et al. 2012a). No

vaccine that achieves complete protection has been discovered, however, the conjugate

vaccine covers more than 85% of tested clinical K. pneumoniae isolates and appears safe

for maternal use (Ahmad et al. 2012b).

1.1.1.2.1 Antibiotic resistance

K. pneumoniae is naturally resistant to aminopenicillins and carboxypenicillins as it

produces a potent β-lactamase (SHV-1) which inactivates the antibiotic (Brisse, Grimont

& Grimont 2006). This activity can in turn be inactivated by clavulanic acid. In recent

years a wider range of resistance to β-lactams has resulted in the use of third generation

cephalosporins, quinolones and aminoglycosides (Brisse, Grimont & Grimont 2006). The

emergence of ESBL K. pneumoniae strains that are also resistant to third generation

antibiotics have increased in frequency and become a cause for concern (Breurec et al.

2013; Yu et al. 2002; Hardy, Legeai & O’Callaghan 1980). In the case of these MDR K.

pneumoniae pathogens, carbapenem antibiotics have been utilised as treatment

options. However, since the first emergence of carbapenem hydrolysing enzymes in

1993, KPC strains have continued to increase globally (Bradford et al. 1997; Ahmad et

7

al. 1999). They are able to hydrolyse penicillins, all cephalosporins, monobactams,

carbapenems, and β-lactamase inhibitors (Papp-Wallace et al. 2010). Colistin and

tigecycline have been reserved for last resort therapy but colistin-resistant K.

pneumoniae strains have also been reported in surveillance studies (Ah, Kim & Lee

2014). These highly resistant pathogens have very few antibiotic treatments left.

1.1.1.3 Epidemiology

Klebsiella species occur naturally and can be found in two different habitats. In the

environment they are present in water, sewage, soil and on plants (Brisse, Grimont &

Grimont 2006). They can also be found on the mucosal surface of humans and animals

(Podschun & Ullmann 1998). In humans, K. pneumoniae are present as saprophytes and

colonise the nasopharynx and intestinal tract (Podschun & Ullmann 1998). They are

considered transient flora on human skin due to the inability to find good growth

conditions, however, carrier rates on the skin increase dramatically in hospital settings

(Podschun & Ullmann 1998). Colonisation rates of Klebsiella species increase in direct

proportion to length of stay in hospitals and hospital personnel have increased Klebsiella

carriage (Casewell & Phillips 1977).

Studies conducted on the distribution of various serotypes among K. pneumoniae

clinical isolates have found that K1 serotypes were mostly isolated from regions of

similar geographical locations. This included Taiwan, China and Japan but very few in

Europe and the United States (Fung et al. 2000). Other studies have found that in

addition to K1 serotypes, K2 serotypes are prevalent in clinical isolates, especially

causing community-acquired liver abscesses. (Fung et al. 2002; Liu, Wang & Jiang 2013).

Developing countries such as South East Asia, have high numbers of community-

acquired K. pneumoniae infections due to the hypervirulent strains present in that area

8

(Siu et al. 2012). In Western countries, community-acquired infections are not as

prevalent, however, nosocomial infections are a major problem (Brisse, Grimont &

Grimont 2006). Since the first emergence of KPC strains, a report conducted by the

World Health Organisation (WHO) in April 2014 found that KPC has spread to all regions

of the world (WHO 2014). KPC is endemic in the USA, South East Asia, parts of South

America and Europe (Figure 1.1) (Munoz-Price et al. 2013).

Figure 1.1 Global distribution of K. pneumoniae carbapenemase strains by

country of origin

KPC-2 and KPC-3 hydrolyse several different classes of β-lactams. Other

carbapenemase types include VIM, OXA-48, or NDM. (Munoz-Price et al. 2013).

9

1.1.1.4 Virulence factors

1.1.1.4.1 Capsular polysaccharides

K. pneumoniae possess a hydrophilic polysaccharide capsule and is classified into

77 serological types (Figure 1.2) (Ørskov & Fife-Asbury 1977). The thick, capsule protects

the bacteria from phagocytosis and bactericidal serum factors (Podschun, Penner &

Ullman 1992; Williams et al. 1983). Some serotypes such as K7 contain a repetitive

sequence of mannose-α-2/3-mannose or L-rhamnose-α-2/3-L-rhamnose which are

recognised by a surface lectin on macrophages that mediates phagocytosis (Athamna et

al. 1991). Macrophages with the mannose-α-2/3-mannose-specific lectin or mannose

receptor recognise K. pneumoniae and are able to ingest and mediate phagocytosis of

the bacteria (Ofek, Goldhar & Keisari 1995). Serotypes such as K2 which lack mannose-

α-2/3-mannose structures are not recognised by macrophages and are able to evade

phagocytosis (Podschun & Ullmann 1998). These serotypes are mostly associated with

invasive infectious diseases (Podschun & Ullmann 1998). When tested via

intraperitoneal injection in mice it was found that K1 and K2 capsular types showed

highest levels of virulence (Mizuta et al. 1983). Hypervirulent K. pneumoniae strains are

associated with heightened virulence due to the acquisition of the rmpA gene, which

partly mediates the increase in capsule production (Patel, Russo & Karchmer 2014).

1.1.1.4.2 Pili

Type 1 pili are the most common pili found in clinical isolates of K. pneumoniae and

are responsible for D-mannose-sensitive haemagglutination. They are associated with

virulence as they facilitate binding of bacteria to mucus or epithelial cells of the urinary,

gastrointestinal and respiratory tracts (Figure 1.2) (Ofek & Beachey 1978; Venegas et al.

1995). Studies conducted in Escherichia coli have found that type 1 pili can also bind to

10

mannosyl-containing glycoproteins in the urine and saliva, providing an explanation to

colonisation of bacteria in the urinary and respiratory tracts (Reinhart, Obedeanu &

Sobel 1990; Babu et al. 1986). When in the host tissue, type 1 pili are no longer of use

to the bacteria as they trigger phagocytosis and intracellular killing of the bacteria. To

counteract this host defence, expression of type 1 pili may be switched off (Brisse,

Grimont & Grimont 2006).

Type 3 pili are referred to as “mannose-resistant Klebsiella-like heamagglutination”

and were first identified in Klebsiella strains (Clegg & Gerlach 1987). They are

characterised by their ability to agglutinate erythrocytes treated with tannic acid in vitro

(Duguid 1959). Type 3 pili can adhere to endothelial and epithelial cells of the respiratory

and urinary tracts (Hornick et al. 1992; Tarkkanen et al. 1997). In vitro studies have found

that they play a role in biofilm formation on abiotic surfaces and thus are capable of

biofilm mediated catheter infections in the urinary system (Di Martino et al. 2003;

Murphy et al. 2013). Type 3 pili also bind to extracellular matrix proteins such as collagen

and promote biofilm formation due to the exposed tissue basement membrane from

device (e.g. catheters) associated tissue damage (Boddicker et al. 2006).

1.1.1.4.3 Siderophores

Iron is essential for the growth of bacteria as it mainly functions as a redox catalyst

of oxygen and electron transport reactions (Braun 2001). Khimji and Miles (1978) found

that when iron was administered to guinea pigs, the infection potential of Klebsiella was

enhanced. Klebsiella species produce two iron-chelating compounds called enterobactin

(also known as enterochelin) and aerobactin (Figure 1.2). These siderophores have high-

affinity to iron, with enterobactin being the main iron uptake system of enterobacteria

(Brisse, Grimont & Grimont 2006). Aerobactin is not as common as enterobactin as only

11

a few strains produce the siderophore (Williams et al. 1987). However, a study

conducted by Nassif and Sansonetti (1986) found that when the aerobactin gene was

cloned from the plasmid of K1 and K2 K. pneumoniae isolates and transferred into a

siderophore negative strain, a marked increase in virulence was observed. This suggests

that aerobactin has a role in virulence but due to its lower affinity to iron in comparison

to enterobactin, the latter is favoured (Podschun & Ullmann 1998). A study conducted

on the secretion of siderophores in hypervirulent strains of K. pneumoniae found that

these strains secreted a 3 to 7 fold higher concentration, in comparison to non-

hypervirulent strains (Russo et al. 2011).

1.1.1.4.4 Serum resistance and lipopolysaccharides

Other virulence factors associated with K. pneumoniae include serum resistance

and lipopolysaccharides (Figure 1.2). The first-line of defence by host immunity is

phagocytosis and the cascade-like activation of bactericidal serum which creates trans-

membranous pores in the outer membrane of Gram-negative bacteria, leading to an

influx of sodium ions and results in lysis of the bacterial cell (Taylor & Kroll 1985; Ramm

et al. 1983). Commensal bacteria are susceptible to this host defence but pathogenic

Gram-negative bacteria have developed serum resistance mechanisms (Olling 1977).

Lipopolysaccharides are composed of lipid A and a side chain called the “O-antigen”.

There are nine O-antigens in K. pneumoniae, with O1 being the most frequent (Hansen

et al. 1999). Lipopolysaccharides activate the cascade-like reaction of bactericidal serum

causing phagocytosis of the bacterial cell. However, it is believed that in K. pneumoniae

the O-antigen side chain length is important in bypassing activation of complement

factor C3b due to steric effects of the long O chain, thus protecting the bacterium from

first-line host defence (Brisse, Grimont & Grimont 2006).

12

Figure 1.2 Schematic diagram of K. pneumoniae virulence factors

(adapted from Podschun & Ullmann 1998).

13

1.2 The genus Burkholderia

The genus was first defined by Yabuuchi et al. (1992) who proposed that seven

species in the genus Pseudomonas homology group II, be placed in a new genus called

Burkholderia to honour the work of bacteriologist, Walter H. Burkholder. The genus

Burkholderia is part of the Burkholderiaceae family and consists of over 30 species

(Coenye & Vandamme 2003). They are motile, aerobic, Gram-negative bacilli and

contain a cell wall like other Gram-negative bacteria (Breed, Murray & Smith 1957).

Burkholderia species can inhabit diverse ecological niches and have been isolated from

soil, plants, water and insects. They are also found in hospital settings, industrial

environments and cause opportunistic infections in humans (Coenye & Vandamme

2003). Several Burkholderia species are of medical interest due to the severe infections

and rapid disease progression of these bacteria. Species of the Burkholderia cepacia

complex have emerged as important pathogens in individuals with cystic fibrosis and

chronic granulomatous disease, causing morbidity and mortality (Isles et al. 1984;

Johnston 2001)

1.2.1 The Burkholderia cepacia complex

B. cepacia was first placed in the genus Pseudomonas and named Pseudomonas

cepacia due to the bacterium’s phenotypic characteristics, which include the utilisation

of carbon and the presence of polar flagella (Vinion-Dubiel & Goldberg 2003). B. cepacia

was first identified by Walter H. Burkholder, who observed that the bacteria were the

causative agent of ‘sour skin’ onion rot (Vinion-Dubiel & Goldberg 2003). Since the

identification of phenotypically similar but genetically distinct B. cepacia isolates, the

group has been divided into at least 17 species called the Burkholderia cepacia complex

(Bcc) (Vandamme et al. 1997; Leitão et al. 2010).

14

Bcc bacteria are non-sporulating and are typically catalase- and oxidase positive

(Govan, Hughes & Vandamme 1996). Three different selective media have been

developed to isolate Bcc based on their utilisation of lactose, sucrose and their

resistance to antibiotics such as polymyxin, gentamicin and vancomycin. These are

Pseudomonas cepacia agar, oxidation-fermentation polymyxin bacitracin lactose agar

and Burkholderia cepacia selective agar (Woods & Sokol 2006). Bcc bacteria can also

grow on non-selective media such as LB media, blood agar plates and tryptic soy media

(Miller, LiPuma & Parke 2002). On agar plates, Bcc colonies can appear matte and dry

for some strains, and shiny, smooth and slightly raised for other strains (Chung et al.

2003). Optimal growth temperatures are between 30 – 37°C (Govan, Hughes &

Vandamme 1996).

The Bcc is categorised in genomovars that are based on the genetic variances of the

bacteria and are given a species name when a distinguishable phenotypic characteristic

is seen (Vandamme et al. 1997; Ursing et al. 1995). Genomovar I is named as the type

species B. cepacia and since then, many genomovars and species have been identified

(Table 1.1).

Table 1.1 Overview of the Burkholderia cepacia complex

Species name Genomovar designation

B. cepacia B. cepacia genomovar I B. multivorans B. cepacia genomovar II B. cenocepacia B. cepacia genomovar III B. stabilis B. cepacia genomovar IV B. vietnamiensis B. cepacia genomovar V B. dolosa B. cepacia genomovar VI B. ambifaria B. cepacia genomovar VII B. anthina B. cepacia genomovar VIII B. pyrrocinia B. cepacia genomovar IX

(Coenye & Vandamme 2003)

15

Burkholderia cenocepacia (previously known as genomovar III) was named by

Vandamme et al. (2003) and is 0.6 to 0.9 by 1.0 to 2.0 µm. Growth can be seen between

25 - 37 °C, with growth at 42 °C being strain dependent. Certain properties of B.

cenocepacia are also strain dependent. These include yellow pigmentation of bacterial

colonies, urease and β-galactosidase activity, nitrate reduction and malonate utilisation

(Vandamme et al. 2003). Oxidase and catalase activity is present as is lecithinase activity.

1.2.1.1 Disease and clinical presentation

Bcc bacteria are opportunistic pathogens that cause infections in

immunocompromised individuals (Speert 2002). They infect the respiratory and urinary

tract leading to bacteraemia, endocarditis, pneumonia, liver abscesses and septic shock

(Mukhopadhyay, Bhargava & Ayyagari 2004; Govan, Hughes & Vandamme 1996). High

fever and chills have also been observed in infected patients (Mukhopadhyay, Bhargava

& Ayyagari 2004). Bcc infections most commonly occur in cystic fibrosis and chronic

granulomatous disease patients (Speert 2002; Govan, Hughes & Vandamme 1996).

Chronic granulomatous disease is a genetic immunodeficiency disorder of leukocyte

function (Heyworth, Cross & Curnutte 2003). In chronic granulomatous disease, the

oxidative function of leukocytes is disabled (Speert 2002). Bcc bacteria are resistant to

non-oxidative means of phagocytosis and thus infections persist, causing a much higher

risk of detrimental and possibly fatal outcome (Speert et al. 1994).

Over the past decade, Bcc bacteria have become particularly virulent pathogens in

cystic fibrosis patients (Speert 2002). It is believed that there is an imbalance in

oxidant/antioxidant in cystic fibrosis lungs, enabling oxidative-killing-resistant bacteria

to survive (Speert 2002). Co-infection with Pseudomonas aeruginosa protects Bcc

bacteria as P. aeruginosa is able to suppress oxidative radicals, providing a niche where

16

replication in the host can occur after the establishment of chronic P. aeruginosa

infection (Speert 2002).

B. cenocepacia is one of the most prevalent pathogens of the Bcc in cystic fibrosis

patients (Sajjan, Keshavjee & Forstner 2004). Symptoms include severe pulmonary

inflammation, necrotising pneumonia and sepsis (Sajjan et al. 2008). Chronic infections

can lead to ‘cepacia syndrome,’ a rapid deterioration in lung function characterised by

progressive pneumonic illness, marked pyrexia and multi-organ failure (Blackburn et al.

2004). Cystic fibrosis patients infected with Bcc bacteria can develop ‘cepacia syndrome’

years after first being colonised, as was seen in a case study of a boy who developed it

nine years after first infection (Blackburn et al. 2004). The control of infection to prevent

exacerbations is critical in avoiding ‘cepacia syndrome’ and the rapid deterioration of

health. Risk factors associated with Bcc colonisation in patients include hospitalisation

where contamination can occur from Bcc-positive patients via contaminated surfaces,

patient-to-patient transmission and on the hands of health care workers, as well as in

the environment and through social contact (Fung et al. 1998).

1.2.1.2 Treatment

Treatment options for Bcc bacteria are limited due to the resistance of a wide range

of antimicrobial agents (LiPuma 2005). Current treatment options are best tackled on a

case by case basis as no optimum treatment for pulmonary exacerbations are known

(Horsley & Jones 2012). The use of combination therapy for synergistic effects is the best

option with the current lack of novel antimicrobials. Common antibiotics used in

combination include meropenem, ciprofloxacin, minocycline, trimethoprim-

sulfamethoxazole, and chloramphenicol (Zhou et al. 2007). It has been observed that

triple combination antibiotics can have a much greater likelihood of bactericidal effects

17

against Bcc and that combinations such as meropenem, tobramycin and another agent

were bactericidal against 81% to 93% of isolates (Aaron et al. 2000). The safety of such

combinations is unknown as limited published reports on the treatment of Bcc in cystic

fibrosis patients are available (Zhou et al. 2007). The end goal for treatment in Bcc

colonised patients however is not to eradicate the infection but to control the infection

and prevent exacerbations that increase inflammation and further deterioration of lung

function (Aaron et al. 2000). Currently no vaccine is available.

1.2.1.2.1 Antibiotic resistance

Bcc bacteria are among some of the most antibiotic resistant pathogens in clinical

laboratories being resistant to a wide range of drugs such as polymyxins,

aminoglycosides and most β-lactams (Figure 1.3) (Drevinek & Mahenthiralingam 2010).

They are capable of developing in vivo resistance through mechanisms of enzyme

inactivation (β-lactamases, aminoglycoside-inactivating enzymes and dihydrofolate

reductase), alteration of drug targets, cell wall permeability and active efflux pumps

(Drevinek & Mahenthiralingam 2010). Bcc limit access of antibiotics to bacterial cells

through three main mechanisms. The lipopolysaccharide found on the bacteria’s cell

wall limits the binding of Bcc pathogens to cationic antibiotics as well as cationic

peptides of the human innate immune system (LiPuma 2007). Porins are hydrophilic

channels on the cell membrane that allow water soluble antibiotics into the cell. In Bcc

bacteria these channels have been found to be impermeable to antibiotics due to low

porin expression and/or because of changes to the porin channel inhibiting antibiotic

uptake (Parr et al. 1987; Aronoff 1988).

Efflux systems can modulate susceptibility to broad-spectrum antibiotics and

specific antimicrobial compounds (Holden et al. 2009). When the epidemic ET12 lineage

18

B. cenocepacia strain was sequenced, it was found that the bacteria had six classes of

efflux system: the major facilitator superfamily, ATP binding cassette family, small multi-

drug resistance family, resistance nodulation division (RND) family, multidrug and toxic

compound extrusion family and the fusaric acid resistance family proteins (Holden et al.

2009). The RND family is notably the best characterised efflux system in the sequenced

strain and was found to confer resistance to chloramphenicol, trimethoprim and

ciprofloxacin (Drevinek & Mahenthiralingam 2010; Nair et al. 2004). In addition to this,

biofilm formation may contribute to antibiotic resistance by limiting drug diffusion

through the biofilm, drug inactivation in the biofilm, the presence of less susceptible

stationary-phase bacteria and the up-regulation of biofilm associated antibiotic

resistance genes (LiPuma 2007).

19

Figure 1.3 Antibiotic resistance mechanisms of the Bcc bacteria

Antibiotic resistance is in all compartments of the bacteria. The outer membrane

efflux pump actively exports chloramphenicol, trimethoprim and quinolones. A

trimethoprim-resistant dihyrodrofolate reductase (DHFR) enzyme has been

detected in some Bcc bacteria. Porin proteins found on the outer membrane do not

allow transport of some antibiotics into the cell. Two β-lactamase mechanisms

inhibit β-lactam activity. Periplasmic β-lactamases degrade activity of the antibiotic

and penicillin-binding proteins (PBP) are less susceptible to their action

(Mahenthiralingam, Urban & Goldberg 2005).

20

1.2.1.3 Epidemiology

Bcc bacteria are incredibly versatile, surviving in harsh and widespread

environments and adapting to nutritional variances (LiPuma 2005). Such environments

include being able to adapt and survive for long periods of time in disinfectants and

anaesthetic solutions in hospitals (Govan, Hughes & Vandamme 1996). Bcc infections

are mostly nosocomial and are thought to cause infection in cystic fibrosis and chronic

granulomatous disease patients via contaminated ultrasound gel, nebulised

medications, nasal spray, hospital water, and lipid emulsion (Hutchinson et al. 2004;

Balkhy et al. 2005; Nasser et al. 2004; Doit et al. 2004; LiPuma 2005). Community-

acquired Bcc infections are rare, although there have been reports from the 1990’s that

have shown disturbing Bcc infections causing fatality in previously healthy individuals

(Govan, Hughes & Vandamme 1996).

Infections caused by specific genomovars are unanimous in the literature. It is seen

that B. cenocepacia (genomovar III-A/III-B) followed by Burkholderia multivorans

(genomovar II) are the most prevalent Bcc infections for cystic fibrosis sufferers. Four

independent studies conducted in the United States of America (USA), Canada, United

Kingdom (UK) and Italy found that B. cenocepacia was the cause of greater than 50% of

infections in the populations studied (Table 1.2) (LiPuma et al. 2001; Speert et al. 2002;

De Soyza et al. 2004; Agodi et al. 2001). Another study conducted in Australia during a

10 year period found that 43.1% of the population was infected with B. cenocepacia

(Table 1.2) (Ramsay et al. 2013). Pope, Short and Carter (2010) found that in New

Zealand, B. multivorans was the most prevalent pathogen with 79.5% of the cystic

fibrosis population studied being infected with the bacteria (Table 1.2). Epidemic B.

cenocepacia strain ET12 and closely phylogenetically related strains were seen in the

21

population studies in USA, Canada, UK and Italy (LiPuma et al. 2001; Speert et al. 2002;

De Soyza et al. 2004; Agodi et al. 2001). The Australia and New Zealand studies found a

prevalence in the Australian epidemic B. cenocepacia strain ST39 (Ramsay et al. 2013;

Pope, Short & Carter 2010).

Table 1.2 Distribution of Bcc isolates from six cystic fibrosis population studies

‘Other’ refers to other species/genomovars in the Bcc and indeterminate isolates.

Prevalence (%)

Species USA (n=606)ˣ

Canada (n=445)ˢ

Italy (n=86)*

UK (n=29)°

Australia (n=65)ˠ

New Zealand (n=39)+

Mean

B. cepacia 2.6 0.2 3.5 0 12.3 0.0 3.1 B. multivorans 37.8 9.7 4.7 38 26.2 79.5 32.7 B. cenocepacia 50.0 82.9 74.4 55 43.1 12.8 53.0 B. stabilis 0.2 3.8 3.5 0 0 5.1 2.1 B. vietnamiensis 5.1 1.6 0 7 4.6 2.6 3.5 Other 4.3 1.8 13.9 0 13.8 0 5.6

ˣ LiPuma et al. 2001, ˢ Speert et al. 2002, *Agodi et al. 2001, ° De Soyza et al. 2004,

ˠ Ramsay et al. 2013, + Pope, Short & Carter 2010

There have been few epidemiological studies on the prevalence of Bcc in chronic

granulomatous disease. Greenberg et al. (2009) found that over an 11 year period, no

epidemic strains infecting multiple patients were identified. The epidemic strains

commonly seen in cystic fibrosis communities such as ET12 were also not observed. In

contrast to cystic fibrosis where B. cenocepacia and B. multivorans dominate infections,

a much broader representation of Bcc have been isolated from the chronic

granulomatous disease study population, including Burkholderia ambifaria and

Burkholderia metallica; pathogens that are rarely isolated from humans, including

patients with cystic fibrosis (Greenberg et al. 2009). The reasons for differences in

22

epidemiology between cystic fibrosis and chronic granulomatous disease are not readily

apparent however it is thought that the interaction between the pathogen and the host

is a factor (Greenberg et al. 2009).

1.2.1.4 Virulence factors of Burkholderia cenocepacia

1.2.1.4.1 Quorum sensing

Quorum sensing in B. cenocepacia allows the bacteria to communicate with one

another and coordinate multicellular behaviour by gene regulation on the basis of

population density (Loutet & Valvano 2010). The cepIR quorum sensing system in B.

cenocepacia mediates the production of N-octanoylhomoserine lactone and it has been

shown that this system is required for motility, biofilm stability and virulence in the rat

agar model of chronic lung infection (Tomlin et al. 2005; Sokol et al. 2003; Loutet &

Valvano 2010). B. cenocepacia has a second homoserine lactone-producing quorum

sensing system labelled cciIR that contributes to the regulation of the same functions as

cepIR though in reverse, suggesting that a complex network of gene regulation occurs in

response to bacterial cell density (Malott et al. 2005; Loutet & Valvano 2010). A third

quorum sensing system exists that can utilise non-homoserine lactone compounds such

as cis-2-dodecenoic acid in a cell density-dependent manner (Boon et al. 2008). This

diffusible signal has many of the same functions as cepIR and cciIR (Loutet & Valvano

2010).

1.2.1.4.2 Siderophores

B. cenocepacia synthesise four siderophores during conditions of iron depletion.

These are ornibactin, pyochelin, cepaciachelin, and cepabactin that act to scavenge free

iron from the surrounding environment (Loutet & Valvano 2010; Drevinek &

23

Mahenthiralingam 2010). The most prominent siderophore produced by most strains of

B. cenocepacia is ornibactin (Darling et al. 1998). Studies have shown that ornibactin is

important in virulence of rat agar bead, G. mellonella, and C. elegans infection models

and that iron may play a role in adherence and colonisation of the bacteria (Sokol et al.

1999). B. cenocepacia can also use ferritin, an iron-binding protein, to serve as an

important iron source during cystic fibrosis lung infections as ferritin is in much higher

concentrations in cystic fibrosis patients than healthy individuals (Whitby et al. 2006).

1.2.1.4.3 Motility and adherence

The flagellum is important to B. cenocepacia as it allows motility and enables the

pathogen to invade the host (Tomich et al. 2002). Drevinek et al. (2008) found that when

B. cenocepacia was incubated in cystic fibrosis sputum, increased transcriptional flagella

genes were detected. Retained motility may account for the pathogen’s ability to invade

host cells and cause severe infection (Drevinek & Mahenthiralingam 2010). It has also

been observed that the regulation of flagellar gene transcription is dependent on

quorum sensing (O’Grady et al. 2009). Certain strains of B. cenocepacia such as ET12

express pili (Urban et al. 2005). Cable pili are peritrichous organelles on the surface of

the bacteria and it has been demonstrated that they are capable of adhering to mucin

as well as human buccal, bronchial, and respiratory epithelial cells (Sajjan et al. 1992;

Sajjan et al. 2000; Chiu et al. 2001). The expression of cable pili in combination with

adhesion AdhA bound to cytokeratin 13 has been shown to cause optimum binding

capabilities and transmigration into the host (Urban et al. 2005). Cytokeratin 13 is a

protein expressed on the basal layer of tracheal and bronchial epithelial cells. When

repeated injury and repair occurs an increase in expression is seen (Urban et al. 2005).

This condition is especially observed in cystic fibrosis patients and allows for a suitable

24

environment for highly transmissible B. cenocepacia strains (Sajjan et al. 2000; Urban et

al. 2005).

Thus far the cable pili is the only adhesin which has been extensively studied in B.

cenocepacia however, recent studies have identified the presence of trimeric

autotransporter adhesins (TAA) (Mil-Homens & Fialho 2011). These adhesins are surface

proteins found in Gram-negative bacteria and have been demonstrated to adhere to

extracellular matrix proteins and host cells (Mil-Homens et al. 2014). TAAs are involved

in biofilm formation, cell-to-cell aggregation, protecting the bacterium from host

immune responses (serum resistance), and promoting the invasion of host cells (Heise

& Dersch 2006; Serruto et al. 2009).

1.2.1.4.4 Polysaccharides

B. cenocepacia express lipopolysaccharides which are one of the main components

of the outer surface of the bacteria (Drevinek & Mahenthiralingam 2010). The O-antigen

contained within the lipopolysaccharide prevents phagocytosis within the host but also

interferes with B. cenocepacia adherence to epithelial cells (Saldías, Ortego & Valvano

2009). Some B. cenocepacia strains do not contain the O-antigen, alluding to the fact

that the O-antigen is not particularly necessary for virulence. B. cenocepacia also

produce exopolysaccharides that are released into the surrounding environment and

help with biofilm formation. Exopolysaccharides give the bacteria a mucoid appearance

and have been associated with the chronicity of infection in cystic fibrosis (Drevinek &

Mahenthiralingam 2010). Expression of exopolysaccharide is strain specific and when

expressed does show an increase in virulence, however, the absence of

exopolysaccharide does not mean the strain is incapable of producing severe infection

in the host and this should not be ruled out (Drevinek & Mahenthiralingam 2010).

25

1.3 Macrophage infectivity potentiator proteins

The highly conserved immunophilin superfamily is a group of proteins that are

found in plants, bacteria, fungi and vertebrates. The superfamily is divided into three

unrelated amino acid sequence subfamilies based on the immunosuppressive

compound they bind: FK506-binding proteins (FKBP) bind to FK506 and rapamycin,

cyclophilins bind to cyclosporine A and parvulins bind to juglone (Göthel & Marahiel

1999). Macrophage infectivity potentiator (Mip) proteins are microbial FKBP’s that

exhibit peptidylprolyl cis/trans isomerase (PPIase) activity (Barik 2006). PPIase activity

is essential in proper protein folding as it accelerates the spontaneous isomerisation of

the cis/trans peptidylprolyl bond (Schmid 1993).

Mip proteins were first identified by Cianciotto et al. (1989) who found a 24-kDa

surface protein on the bacterial species Legionella pneumophila (the causative agent of

Legionnaires' disease), involved in the intracellular virulence of the pathogen. The

construction of an L. pneumophila mutant which was defective in the expression of the

24-kDa protein showed an impairment in the pathogens ability to infect U937 cells and

human alveolar macrophages but regained infectivity when the 24-kDa gene was intact,

suggesting that the protein is needed for full virulence of L. pneumophila (Cianciotto et

al. 1989). Studies conducted by Fischer et al. (1992) found that the Mip protein of L.

pneumophila had PPIase activity, and that it was inhibited by FK506 and was resistant

to cyclosporin A; reminiscent of members in the FKBP family. Mip proteins also have

homologous regions similar to those found in FKBP’s of eukaryotic organisms (Fischer et

al. 1992). Thus, Mips represent a bacterial gene product of the FKBP subfamily and

shares similar characteristics to eukaryotic proteins (Fischer et al. 1992).

26

Since the first discovery of the Mip protein in L. pneumophila, other Mip-like

proteins have been identified in different bacteria including Chlamydia trachomatis,

Neisseria gonorrhoeae, Burkholderia pseudomallei and the protozoan Trypanosoma

cruzi (Lundemose, Kay & Pearce 1993; Leuzzi et al. 2005; Norville et al. 2011a; Moro et

al. 1995). Similar to the L. pneumophila study, Leuzzi et al. (2005) created a knockout

strain of N. gonorrhoeae Mip protein by deleting the Mip gene. It was found that the

Mip mutant strain had the same adherence and internalisation capabilities in

macrophages as the wild-type strain. However a reduced survival 24 hours following

infection was observed, suggesting that Mip proteins in N. gonorrhoeae is important in

the persistence of the pathogen in macrophages and protecting the pathogen from

macrophage mediated killing (Leuzzi et al. 2005). Similarly Norville et al. (2011a)

identified a Mip protein in B. pseudomallei. Deletion of the Mip gene resulted in reduced

intracellular survival and replication in eukaryotic cells, and attenuation in BALB/c

mouse models of infection. The C. trachomatis Mip protein was identified by inhibiting

the protein with rapamycin; confirming that it is part of the FKBP family (Lundemose,

Kay & Pearce 1993). Based on the results of rapamycin inhibition, it was suggested that

inhibiting the protein interfered with one or more early stage infective events of

intracellular infection (Lundemose, Kay & Pearce 1993). As observed, Mip proteins are

a virulence factor for a number of pathogenic bacteria, as interfering with this protein

leads to a decrease in infectivity of the pathogen. Due to the highly conserved nature of

Mips, it is believed that putative Mip proteins exist in K. pneumoniae and B. cenocepacia.

Therefore, they have been presented as potential novel antimicrobial targets.

27

1.4 Virulence targets for novel inhibitors

Targeting virulence factors found in a wide range of bacteria can offer potential for

the development of novel inhibitors with broad-spectrum activity (Sarkar-Tyson &

Atkins 2011). Targets need to be specific to bacteria with no human homologs and be

essential for bacterial virulence (Sarkar-Tyson & Atkins 2011). Developing novel

inhibitors to treat MDR pathogens can greatly improve prognosis, economic burden and

healthcare among many other issues associated with difficult to treat infections.

Decreasing the infectious potential of these pathogens with novel inhibitors can

improve antibiotic activity in killing the bacteria and minimise the likelihood of the

development of MDR strains. Examples of novel inhibitors include quorum sensing

inhibitors, cell division inhibitors and Mip inhibitors.

1.4.1 Quorum sensing inhibitors

Interfering with the quorum sensing system and the activation of quorum sensing

genes affects bacterial virulence. Quorum sensing inhibitors work by either inhibiting

the signal molecules produced by bacteria or interfering with the signal receptor

(Rasmussen & Givskov 2006). Signal molecule inhibition can be achieved through

chemical degradation, enzymatic destruction and metabolism of the N-Acyl homoserine

lactone molecule, whereas signal receptor interference can be achieved through the

blockage or destruction of receptor protein (Rasmussen & Givskov 2006). Quorum

sensing inhibitors can be synthetic in origin, however, inhibitors are found widely in

nature. Plants, fungi and animals have co-existed with bacteria for many years and some

produce quorum sensing inhibitors to reduce colonisation capability and competition

(Rasmussen & Givskov 2006). An example of quorum sensing inhibition is explored by

O’Loughlin et al. (2013) who found that the use of the compound meta-bromo-

28

thiolactone inhibited the expression of the genes encoding the virulence factor

pyocyanin, preventing biofilm formation and protected human lung cells from killing by

P. aeruginosa; a species commonly co-infecting cystic fibrosis patients with the Bcc

pathogens.

1.4.2 Cell division inhibitors

All bacterial species possess mechanisms of cell division as it is essential for

propagation (Sarkar-Tyson & Atkins 2011). The FtsZ protein is found in almost all

bacterial species and forms the contractile ring called the Z ring on the inner surface of

the cytoplasmic membrane at the site of division (Buddelmeijer & Beckwith 2002). Like

tubulin in eukaryotic cells, FtsZ has GTPase activity and forms tubulin-like protofilaments

(Boer, Crossley & Rothfield 1992; Erickson et al. 1996). The differences between

eukaryotic and prokaryotic tubulin has enabled exploitation to find compounds that

inhibit bacterial tubulin and not affect host tubulin (Buddelmeijer & Beckwith 2002).

Inhibition of the FtsZ protein can be through natural means or synthetic. Screening

of FtsZ inhibitors in extracts of microbial fermentation and plants found that viriditoxin

selectively inhibited FtsZ polymerization and GTPase activity in vivo and in vitro without

being toxic to eukaryotic cells (Wang et al. 2003). Other natural sources of FtsZ inhibition

are polyphenols called zantrins which inhibit protein polymerisation by either

destabilising FtsZ polymers or by stabilising FtsZ profilaments and altering Z ring

assembly (Margalit et al. 2004; Buddelmeijer & Beckwith 2002). Another selective

inhibitor which was developed by Läppchen et al. (2005) called 8-bromoguanosine 5’-

triphosphate was based on the structure of the natural substrate GTP. This compound

had specific competitive inhibition of FtsZ polymerization and GTPase activity.

29

1.4.3 Mip inhibitors

Juli et al. (2011) looked at various compounds to identify new small-molecule

inhibitors of Mip by starting with known human T-cell FKBP (FKBP12) ligands (substance

A and B) to test in L. pneumophila. These ligands bear a pipecoline moiety and so

resemble the rapamycin anchoring group, but lack the macrocyclic portion thereby

cancelling the immunosuppressive action. Out of the two substances, B, showed a good

fit of the pipecoline-sulfonamide anchor group and was approached for synthesis into

benzylsulfonamide. Experiments were conducted to test the interaction of the

compound with Mip proteins and strong binding was observed (Juli et al. 2011). When

the substance was tested on invasion and intracellular infection it was found that the

substance did not influence replication of the wild-type in human macrophage-like U937

cells. In contrast rapamycin treated wild-type L. pneumophila were not able to replicate

in the cells or were degraded (Juli et al. 2011). These results suggest that Mip proteins

are needed for virulence, however, PPIase activity may not be necessary for virulence.

The target molecule for Mip mediated PPIase activity is still unknown and therefore,

testing substance B once the target molecule has been identified will be more insightful

(Juli et al. 2011).

Similar to the L. pneumophila Mip inhibitor study, Begley et al. (2014) examined

pipecolic acid derived compounds in B. pseudomallei. A wide selection of derivatives

were tested which mimicked the pipecoline group of rapamycin and it was found that

the racemic compounds 37 and 183 inhibited PPIase activity at low micromolar

concentrations. The S enantiomer compound 168 and racemic compound 40 inhibited

PPIase activity at high levels (Begley et al. 2014). When tested in a macrophage-based

cytotoxicity assay, it was found that compounds 40, 160 and 183 significantly reduced

30

cytotoxicity by 30 – 40%. Compound 37 had decreased inhibitory activity however was

higher than the control experiment (Begley et al. 2014). When tested in macrophage

only controls, no cytotoxicity was observed for compounds 37 and 183, and less than

10% cytotoxicity in compounds 40 and 160. Thus, the minimal cytotoxicity observed in

non-infected macrophages provides evidence that this compound series has little

adverse effect on the biological function of healthy mammalian macrophages in vitro

and has great potential for drug development into novel inhibitors which target the Mip

protein (Begley et al. 2014).

1.5 In vitro models for novel inhibitor testing

Before inhibitors can be tested in a clinical setting, extensive pre-clinical research

must be conducted to assess the efficacy and safety of novel inhibitors. Many in vivo

models exist to study host-microbe interactions and inhibitor testing. For example,

insect models e.g. Galleria mellonella, zebrafish model and animal models such as mice.

However, few in vitro models exist. In vitro models provide key aspects of infection and

disease process in a physiologically relevant manner and simplify the process for

analytical studies (MacGowan, Rogers & Bowker 2001; Crabbé, Ledesma & Nickerson

2014). In vitro models are cost efficient and are less likely to raise ethical issues

(Ramarao, Nielsen-Leroux & Lereclus 2012). As models need to be optimised for each

individual bacterial species, model design is necessary to accurately test the efficacy of

novel inhibitors. Some in vitro models that do exist include biofilm models, three-

dimensional (3D) tissue-engineered models and cell based models. These models

provide a method by which to test the efficacy of novel inhibitors before using the more

expensive in vivo infection models.

31

1.5.1 Biofilm models

Biofilm growth models can be classified into two systems: open and closed. Closed

models have the advantage of being simple and applicable in high-throughput analysis

(Lourenço et al. 2014). An example of a closed system model is by using a micro titre

plate such as a 96-well plate to inoculate bacterial culture and incubate at appropriate

conditions for 24-48 hours. Biofilms form as a ring around the wells and washing the

wells remove planktonic cells (Christensen et al. 1985). The remaining biofilm can be

stained with crystal violet and dissolved in acetone to quantify biomass by measuring

optical density (Christensen et al. 1985). This method is rapid and reproducible,

however, the crystal violet stains both viable and dead cells and therefore the

relationship between biomass and biofilm viability is unknown (Peeters, Nelis & Coenye

2008).

Open biofilm systems replicate in vivo conditions by controlling nutrient delivery,

flow and temperature (Macià, Rojo-Molinero & Oliver 2014). The flow cell method

utilises a vessel with sterile broth culture that provides medium through a peristaltic

pump (Klausen et al. 2003; Nielsen et al. 2011). Bacteria are then directly inoculated into

the flow cells by injection through silicone tubing. The cells are then attached to a

surface such as a transparent, non-fluorescent microscope coverslip where biofilm

formation can occur (Klausen et al. 2003; Nielsen et al. 2011). This process allows for a

thicker biofilm however takes several days to prepare (Nielsen et al. 2011).

1.5.2 3D tissue-engineering models

3D tissue-engineered models consist of multiple cell types and a naturally formed

extracellular matrix that mimic in vivo disease processes (Shepherd et al. 2009). 3D

32

tissue is engineered by seeding human skin (keratinocytes and fibroblasts) onto a

decellularised dermis that is treated to retain its native basement membrane. After 10-

14 days of culture the reconstituted tissue resembles characteristics of normal human

skin and can be infected with bacterial culture (Shepherd et al. 2009). Skin constructs

can then be homogenised to determine viable bacterial counts or fixed in formalin for

histological analysis (Shepherd et al. 2009). 3D tissue-engineered models provide a

physiological similar structure to normal tissue and mimics the functions and responses

of these tissues. However, a disadvantage of this model is the long culture time to form

the tissue when comparing to two-dimensional cell monolayers (Mazzoleni, Di Lorenzo

& Steimberg 2009).

1.5.3 Cell based models

Cell based models consist of a confluent monolayer of cells which are then infected

with bacteria to determine virulence of the pathogen. Cell based models were used for

testing the Mip inhibitors in L. pneumophila and B. pseudomallei (Juli et al. 2011; Begley

et al. 2014). In the L. pneumophila Mip inhibitor study, macrophages were infected with

bacteria in the presence of inhibitor and then extracellular bacteria were killed to

enumerate intracellular bacteria only. Intracellular bacteria were then plated out on

agar plates to determine colony-forming unit (CFU) counts, which is a measure of viable

bacteria, to determine if the Mip inhibitors had an effect on internalisation and

replication of the pathogen (Juli et al.2011).

In the B. pseudomallei study, cytotoxicity assays were performed to assess the

effects Mip inhibitors had on intracellular replication of the pathogen (Begley et al.

2014). Confluent macrophage monolayers were infected with bacteria that had been

pre-treated with Mip inhibitors. After infection, bacteria were removed and media was

33

added supplemented with antibiotic and inhibitor before incubation for 24 hours. After

24 hours, cytotoxicity was measured by measuring lactate dehydrogenase release

(Begley et al. 2014). Changes in lactate dehydrogenase release provided insight on the

effects Mip inhibitors had on intracellular replication of B. pseudomallei. Cell based

models are cost efficient and relatively easy to conduct making them attractive models

for pre-clinical studies (Zang et al. 2012).

1.6 Project aim

This project aims to develop in vitro model methods, to test the potential broad-

spectrum activity of Mip inhibitors in K. pneumoniae and B. cenocepacia. To accomplish

this aim, four objectives will be met.

The first objective is to identify the homology between the K. pneumoniae Mip and

the B. cenocepacia Mip against the L. pneumophila and B. pseudomallei Mip. To

investigate this, online tools such as NCBI and ExPASy will be utilised. A multiple

sequence alignment will then be created to compare the sequences of all four

pathogens.

The second and third objectives are to develop cell infection assay models to test

the Mip inhibitors against adherence, and internalisation, survival and replication of the

pathogens. Two methods will be developed. The first method will be to test the

adherence capabilities of the bacterial species by challenging macrophages with K.

pneumoniae and B. cenocepacia and counting the number of bacteria that were able to

adhere to the macrophages via measurable plate counts. The second method will be to

test the internalisation, survival and replication of K. pneumoniae and B. cenocepacia by

challenging macrophages with K. pneumoniae and B. cenocepacia and counting the

34

number of bacteria that were able to internalise, survive and replicate in the

macrophages via measurable plate counts at 0 hour and 24 hour post-infection.

The final objective will be to use an optimised adherence assay and internalisation,

survival and replication assay method to test the efficacy of the Mip inhibitors for K.

pneumoniae and B. cenocepacia. By observing any changes in adherence and

internalisation of bacteria in macrophages we can identify if inhibition of the Mip protein

impacts virulence of the pathogens.

1.7. Significance

K. pneumoniae and B. cenocepacia are both MDR pathogens of public health

interest that cause incredibly difficult to treat infections and increased morbidity and

mortality rates. MDR strains of these bacteria are increasing in prevalence and some

strains have become resistant to all current available first-line drug therapies. Research

into common virulence targets of bacteria have become an attractive area of study as

novel inhibitors is urgently needed. Mip proteins are part of the FKBP family and are

encoded by pathogenic bacteria. They are involved in intracellular virulence of bacterial

species and inhibition of the Mip protein via knock-out mutants or inhibitors have shown

a decrease in virulence. This project will develop models to test the efficacy of Mip

inhibitors and determine an optimised way of measuring the impact they have on

interfering with virulence and replication. This project will also broaden the current

knowledge available on Mip proteins and the effect Mip inhibitors have on virulence in

K. pneumoniae and B. cenocepacia. By increasing our knowledge on Mip inhibition we

can see whether these inhibitors have potential as broad-spectrum antibacterial agents.

35

Chapter Two

Materials and methods

36

2. Materials and methods

2.1 Materials

2.1.1 Bacterial strains and mammalian cell line

K. pneumoniae strains used in this study are summarised in Table 2.1 and B.

cenocepacia strains used in this study are summarised in Table 2.2 All strains were

provided by Dr. T. J. J. Inglis, PathWest Laboratory Medicine WA, J block, QEII Medical

Centre, Nedlands. The mammalian cell line used in this study was RAW264.7 mouse

macrophage cells, provided by Professor Xu, School of Pathology and Laboratory

Medicine, University of Western Australia.

Table 2.1 K. pneumoniae strains used in this study

Table 2.2 B. cenocepacia strains used in this study

K. pneumoniae

strain

Capsular

type

Produce mucoid

colonies on agar

plates

Clinical

isolate

Multi-drug

resistant

MGH78578 K52 No No Yes

ST23.1 K1 Yes Yes No

ST23.2 K1 Yes Yes No

ST86 K2 Yes Yes No

ST628 Unknown No Yes No


ST14 K2 No Yes No


B. cenocepacia strain

Genomovar and lineage

Clinical Isolate

Multi-drug resistant

164 III-B Yes No

165 III-B Yes No

167 III-C Yes No

37

2.1.2 Growth media and supplements

Media used to grow the bacterial strains and RAW264.7 macrophage cells are

summarised in Table 2.3. LB broth was stored at room temperature. LB agar plates,

blood agar plates, Dulbecco’s Modified Eagle Medium (DMEM) and Leibovitz’s L-15

Medium (L-15) were stored in the fridge at 4 °C. Supplements were added to media as

required. GlutaMAXTM was stored in the fridge at 4°C. Foetal bovine serum (FBS) was

aliquoted into 50 mL tubes and stored in the freezer at -20 °C. Supplements were added

to DMEM by adding 10% FBS, 1% PenStrep and 1% GlutaMAXTM. Supplements were

added to L-15 by adding 10% FBS.

Table 2.3 Media and supplements used in this study

2.1.3 Antibiotics and chemicals

Antibiotics were added to media as required. Concentrations and preparation of

antibiotics used for experiments are listed in Table 2.4. PenStrep was stored as 5 mL

Media and supplements Supplier

LB broth PathWest

LB agar plates PathWest

Blood agar plates PathWest

DMEM Gibco by Life Technologies (USA)

L-15 Gibco by Life Technologies (USA)

GlutaMAXTM Gibco by Life Technologies

FBS Gibco by Life Technologies

38

aliquots and kanamycin was filter sterilised using a 0.2 μm filter and stored as 1 mL

aliquots. All antibiotics were stored in the -20 °C freezer.

Chemicals used during the experiments are listed in Table 2.4. Prepared 1x

phosphate-buffered saline (PBS) was autoclaved at 121 °C for 15 min, cooled and stored

at room temperature. Triton-X was filter sterilised using a 0.2 μm filter and stored at

room temperature. Dimethyl sulfoxide (DMSO) was stored at room temperature. Pre-

made PBS, GlutaMAXTM and TrypLETM were stored in the fridge at 4 °C and cytochalasin-

D was stored as 50 μL aliquots in the -20 °C freezer.

Table 2.4 Antibiotics and chemicals used in this study

Antibiotics and chemicals Composition Supplier

PenStrep N/A Gibco by Life Technologies (USA)

Kanamycin (50 mg/mL) 1g dissolved in 20mL dH2O Sigma-Aldrich chemical company (USA)

Pre-made PBS N/A Gibco by Life Technologies (USA)

PBS

10mM disodium phosphate, 156mM sodium chloride,

2mM monopotassium phosphate

Invitrogen by Life Technologies (USA)

Cytochalasin-D (1 mg/mL) 1mg dissolved in 1mL DMSO Sigma-Aldrich chemical company (USA)

Triton-X (1%) 500μL dissolved in 50mL PBS Sigma-Aldrich chemical company (USA)

TrypLETM N/A Gibco by Life Technologies (USA)

DMSO N/A Sigma-Aldrich chemical company (USA)

Mip inhibitor 354 Dissolved in DMSO to a final concentration of 5mM

Professor Ulrike Holzgrabe, Würzburg University (Germany)

Mip inhibitor 214 Dissolved in DMSO to a final concentration of 5mM

Professor Ulrike Holzgrabe, Würzburg University (Germany)

39

2.1.4 Bioinformatic tools and software

The Basic Local Alignment Search Tool (BLAST) from the National Centre for

Biotechnology Information (NCBI) was utilised to identify Mip homologues in K.

pneumoniae and B. cenocepacia. ClustalW-PBIL in ExPASy was used to create a multiple

sequence alignment. GraphPad Prism 6 was used for statistical analysis and Excel 2013

was used to create graphs and determine standard errors of the mean (SEM).

2.2. Methods

2.2.1 Bacterial recovery from glycerol stock and maintenance

Bacterial strains were recovered by taking a loop full of glycerol bacterial stock

stored in the 80 °C freezer and streaking onto blood agar plates. Plates were incubated

under aerobic conditions at 37 °C overnight for K. pneumoniae and 48 hours for B.

cenocepacia. After incubation, plates were stored in the fridge at 4 °C. Fresh blood agar

plates were inoculated with glycerol bacterial stock every 2-3 weeks.

2.2.2 Mammalian tissue culture

2.2.2.1 Cell revival

A vial containing RAW264.7 macrophage cells was rapidly thawed from storage in

the -80 °C freezer and 1 mL of DMEM was added to the vial. The suspension was then

transferred to a Sarstedt 15 mL tube and centrifuged at 423 x g for 5 min. The

supernatant was discarded and 1 mL of DMEM was added to the cell pellet to resuspend.

The 1 mL resuspension was then added to a culture flask containing 9 mL DMEM to make

up a final volume of 10 mL. The flask was incubated at 37 °C with 5% CO2.

40

2.2.2.2 Cell passage and maintenance

Confluent cell cultures were diluted into new culture flasks. The media was

discarded and 5 mL of PBS was added to the flask to wash the cells. The PBS was then

discarded and 5 mL TrypLETM was added to the flask and incubated at 37 °C for 15 min

to allow dissociation of the cells. After incubation if the cells had not dissociated a

scraper was used to remove all remaining adherent cells. The suspension was then

transferred into a 15 mL tube and a 5 mL aliquot of DMEM was added to the cell

suspension to deactivate the TrypLETM reaction. The cell suspension was then

centrifuged at 423 x g for 5 min. The supernatant was discarded and 1 mL of DMEM was

added to the 15 mL tube to resuspend the cell pellet. Required amount of resuspended

cells was then added to a new flask with DMEM to make up a final volume of 10 mL.

2.2.3 Cell infection assays

2.2.3.1 Cell preparation for in vitro assays

RAW264.7 macrophage cells were prepared for infection assays by following the

method outlined in section 2.2.2.2 until the centrifugation step. Cells were then diluted

in DMEM and counted using a haemocytometer. Cells were made up to a concentration

of 4 x 105 cells/mL and seeded onto Costar® 24-well plates by adding 1 mL of cell

suspension to each well. The 24-well plates were then incubated overnight at 37 °C with

5% CO2, achieving a confluent monolayer of 1 x 106 cells/mL the following day.

2.2.3.2 Bacterial growth for in vitro assays

Overnight cultures were prepared by inoculating 10 mL LB broth in a Sarstedt 30

mL tube with a single bacterial colony from blood agar plates (section 2.2.1) and

incubating overnight under aerobic conditions at 37 °C with agitation.

41

2.2.3.4 Dilution of overnight bacterial culture for enumeration and MOI calculations

Overnight bacterial culture was prepared as outlined in section 2.2.3.2. The

overnight culture was then diluted into LB broth to make up a final volume of 10 mL

according to the following dilutions and vortexed in 30 mL tubes (Table 2.5).

Table 2.5 Dilutions of overnight culture in media

Neat tube Overnight culture Media Dilution

1 500μL 9500μL 1 in 20

2 1000μL 9000μL 1 in 10

3 1500μL 8500μL 3 in 20

Each 30 mL tube was labelled neat and was then serially diluted (1 in 10) from 10-1

to 10-7 into LB broth in 24-well plates. Overnight culture, neat dilutions and subsequent

dilutions were then measured using a spectrophotometer (Biochrom WPA CO7500

Colorimeter) at an optical density of 600 nm. On LB agar plates, serial dilutions 10-6 and

10-7 for each neat dilution were spread plate 3x with 100 μL aliquots of bacterial

suspension and incubated overnight for K. pneumoniae and 48 hours for B. cenocepacia

under aerobic conditions at 37 °C. Colonies were counted on each plate to determine

CFU/mL (Appendix). Each experiment was repeated three times.

2.2.3.4.1 Overnight bacterial culture dilutions for in vitro assays

Based on the results of the dilution experiments, overnight bacterial culture

(section 2.2.3.2) was diluted in L-15 media until an absorbance reading of 0.25-0.35 was

reached (depending on strain) for K. pneumoniae and 0.10-0.20 for B. cenocepacia for

each in vitro assay conducted. This dilution was labelled as neat. The neat bacterial

cultures were then serially diluted in L-15 (1 in 10) from 10-1 to 10-7. Serial dilutions 10-6

42

and 10-7 were spread plate 3x with 100 μL aliquots of bacterial suspension on LB agar

plates and incubated overnight for K. pneumoniae and 48 hours for B. cenocepacia

under aerobic conditions at 37 °C. The following day bacterial colonies were counted to

determine CFU counts (Appendix).

2.2.3.4 In vitro assays

2.2.3.4.1 Adherence assays

RAW264.7 macrophage cells were prepared the day before as outlined in section

2.2.3.1. Overnight bacterial cultures were prepared as outlined in section 2.2.3.2. Dilute

bacterial suspensions in L-15 media were prepared as outlined in section 2.2.3.3.1. The

DMEM in the 24-well plates containing the RAW264.7 macrophage cells was discarded

and 1 mL aliquots of L-15 media with 1 μg/mL cytochalasin-D was added to all wells and

incubated at 37 °C under aerobic conditions for 30 min. Prior to infection, 1 μg/mL

cytochalasin-D was added to dilute bacterial suspensions in L-15 media without

incubation. After the 30 min incubation, the L-15 media with cytochalasin-D was

discarded and 1 mL aliquots of dilute cytochalasin-D treated bacterial suspensions were

added to wells containing pre-treated macrophages at an approximate multiplicity of

infection (MOI) of 1:100 for K. pneumoniae and 1:200 for B. cenocepacia. For the

controls, 1 mL aliquots of dilute cytochalasin-D treated bacterial suspensions were

added to wells containing no macrophages. The 24-well plates were then incubated at

37 °C under aerobic conditions for 1 hour. After incubation, bacterial suspensions were

discarded from all wells, and the cells were subsequently washed 3x with 1 mL aliquots

of PBS to remove non-adhered bacteria. Cells were subsequently lysed by adding 1 mL

aliquots of 1% Triton X in PBS into all wells and incubated at 37 °C under aerobic

conditions for 15 min. After incubation, wells were scraped and serially diluted (1 in 10)

43

in PBS. Each dilution was plated out on LB agar by dividing the agar plates into thirds and

dropping three individual 100 μL aliquots of bacterial suspension onto each area (three

drop method). Plates were then left to dry at room temperature and incubated

overnight for K. pneumoniae and 48 hours for B. cenocepacia at 37 °C under aerobic

conditions. The following day bacterial colonies were counted to calculate the

concentration at which the bacteria adhered to the macrophages (Appendix).

2.2.3.4.2 Internalisation, survival and replication assays

RAW264.7 macrophage cells were prepared the day before as outlined in section

2.2.3.1. Overnight bacterial cultures were prepared as outlined in section 2.2.3.2. Dilute

bacterial suspensions in L-15 media were prepared as outlined in section 2.2.3.3.1. The

DMEM in the 24-well plates containing the RAW264.7 macrophage cells was discarded

and 1mL aliquots of dilute bacterial suspensions were added to the wells containing the

macrophages at an MOI of 1:1000 for K. pneumoniae and 1:200 for B. cenocepacia. For

the controls, 1 mL aliquots of dilute bacterial suspensions were added to wells

containing no macrophages. The 24-well plates were then incubated at 37 °C under

aerobic conditions for 1 hour. After incubation, bacterial suspensions were discarded

from all wells and were subsequently washed 3x with 1 mL aliquots of PBS to remove

extracellular bacteria. A 1 mL aliquot of 1 mg/mL kanamycin in L-15 media was then

added to each well to kill any remaining extracellular bacteria and plates were incubated

at 37 °C under aerobic conditions. After 1 hour incubation, 1 mL aliquots of 0.01% Triton

X were added to all wells of the 0 hour time point and incubated at 37 °C under aerobic

conditions for 15 min to lyse the macrophages. For the 24 hour time point, 1 mL aliquots

of 250 μg/mL maintenance kanamycin in L-15 media was added to all wells and

incubated at 37 °C under aerobic conditions until time point. After the 15 min

44

incubation, wells were scraped and serially diluted in PBS. Each dilution was plated out

on LB agar using the three drop method (section 2.2.3.4.1). Plates were then left to dry

at room temperature and incubated overnight for K. pneumoniae and 48 hours for B.

cenocepacia at 37 °C under aerobic conditions. At the 24 hour time point, the L-15 media

containing the maintenance kanamycin in each well was discarded. Triton X was then

added to all wells to lyse the macrophage cells and wells were serially diluted and plated

out on LB agar plates as mentioned above. Bacterial colonies were counted to calculate

the concentration at which the bacteria internalised into the macrophages (Appendix).

2.2.3.4.3 Mip inhibitor testing in adherence assays, and internalisation, survival and

replication assays

When testing the Mip inhibitors an extra step was added to incorporate the

inhibitors into the assays. Before commencement of assays, dilute bacterial suspensions

were dispensed in the required volume and incubated with each of the Mip inhibitors

354 or 214 at a concentration of 5 mM at room temperature for 1 hour. For the DMSO

controls, dilute bacterial suspensions in L-15 were incubated with DMSO at a

concentration of 5 mM at room temperature for 1 hour. For the macrophage cell plus

bacteria controls and the bacteria only controls, dilute bacterial suspensions were left

at room temperature without any additives for 1 hour. The methods then followed as

described in section 2.2.3.4.1 for the Mip inhibitor adherence assays and section

2.2.3.4.2 for the Mip inhibitor internalisation, survival and replication assays.

45

Chapter Three

Results of Objective 1

Bioinformatic confirmation of putative Mip homologues in

K. pneumoniae and B. cenocepacia, and quantitative

determination of MOIs

46

3. Bioinformatic confirmation of putative Mip homologues in K. pneumoniae and B.

cenocepacia, and quantitative determination of MOIs

3.1 Strains

The K. pneumoniae and B. cenocepacia strains used in this study are outlined in

Table 2.1 and Table 2.2 respectively, of the materials section. K. pneumoniae strains

were chosen because of their varying capsular types. B. cenocepacia strains were picked

out of a variety of Bcc strains as it has been identified as the most prevalent pathogen

of cystic fibrosis populations.

3.2 Identification of Mip homologues in K. pneumoniae and B. cenocepacia through

bioinformatic analysis

The B. pseudomallei Mip sequence was used to search for homologues using the

NCBI BLAST search tool. The homology was then compared between the putative K.

pneumoniae Mip and B. cenocepacia Mip with the L. pneumophila and B. pseudomallei

Mip. It was found that K. pneumoniae shared 41% and 52% with the L. pneumophila and

B. pseudomallei Mip respectively and that B. cenocepacia shared 40% and 95%

homology with the L. pneumophila and B. pseudomallei Mip respectively. Using the K.

pneumoniae strain MGH78578 sequence (accession number ABR79951.1) and the B.

cenocepacia sequence (accession number WP_009695294.1), a multiple sequence

alignment was created with the online tool ClustalW-PBIL in ExPASy (Figure 3.1). Many

regions of the sequence alignment were highly conserved between all four pathogens,

especially the drug binding domains aspartic acid44 and tyrosine89 previously identified

when the key active site of B. pseudomallei Mip was examined with nuclear magnetic

resonance spectroscopy (NMR) and X-ray crystallography (Norville et al. 2011b).

Aspartic acid44 and tyrosine89 are shown in the boxed areas.

47

Figure 3.1 Alignment of the B. pseudomallei, B cenocepacia, K. pneumoniae and L.

pneumophila Mip sequence

K. pneumoniae shared 52% homology and 41% homology against the B. pseudomallei and

L. pneumophila Mip sequence respectively. B. cenocepacia shared 95% homology and

40% homology between the B. pseudomallei and L. pneumophila Mip sequence

respectively. An asterisk (*) denotes a position with a single fully conserved residue, a

colon (:) denotes conservation among groups of strongly similar properties and a full stop

(.) denotes conservation among groups of weakly similar properties. Red highlighted

residues indicate small + hydrophobic, blue highlighted indicates acidic, green highlighted

indicate hydroxyl + sulfhydryl + amine + G. The boxed areas denote the key active site

residues aspartic acid44 and tyrosine89.

B. pseudomallei B. cenocepacia K. pneumoniae L. pneumophila





48

3.3 Overnight dilution experiments

The concentration of overnight bacterial culture was enumerated in order to

calculate the MOI, which is the ratio in which the macrophages were infected with

bacteria for each assay as a method of standardisation, as described in section 2.2.3.4.

To determine the concentration of overnight bacterial cultures, cultures were diluted

into three known dilutions of 1 in 20, 1 in 10 and 3 in 20. These dilutions were then

serially diluted in LB broth and read by spectrophotometer at 600nm. Serial dilutions

10-6 and 10-7 were spread plate onto agar plates for CFU counts. The CFU counts obtained

the following day for K. pneumoniae and 48 hours for B. cenocepacia were then

calculated to determine CFU/mL of overnight culture.

3.3.1 K. pneumoniae dilution

Strains MGH78578 and ST23.1 were used to determine the overnight concentration

of K. pneumoniae cultures. Due to time constraints, all strains could not be tested,

however, it was found that the K. pneumoniae strains had overnight concentrations

ranging from approximately 1 x 1010 – 2.5 x 1010 and therefore, the same dilution was

sufficient for each strain. It was found that the average overnight concentrations of

MGH78578 and ST23.1 were 2.23 x 1010 CFU/mL and 1.19 x 1010 CFU/mL respectively.

To calculate the MOI needed for each infection assay, the concentration of bacteria

needed from overnight culture was 1 x 109 - 2 x 109 CFU/mL. It was found that a 1 in 10

dilution gave the most accurate concentration range needed, when diluted to an OD600

of between 0.25 and 0.35 for both strains (Figure 3.2 and 3.3).

49

Figure 3.2 Dilution of K. pneumoniae strain MGH78578 overnight cultures

Overnight concentration of strain MGH78578 cultures were an average of 2.23 x

1010 CFU/mL and reached an OD600 of approximately 1.96 (not shown in graph).

Figure 3.3 Dilution of K. pneumoniae strain ST23.1 overnight cultures

Overnight concentration of strain ST23.1 cultures were an average of 1.19 x 1010

CFU/mL and reached an OD600 of approximately 1.93 (not shown in graph).

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

4.00E+09

4.50E+09

0 0.2 0.4 0.6

CFU

/mL

OD600

1 in 20 1 in 10 3 in 20

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

0 0.1 0.2 0.3 0.4 0.5 0.6

CFU

/mL

OD600

1 in 20 1 in 10 3 in 20

50

3.3.2 B. cenocepacia dilution

Strains 164 and 165 were used to determine the overnight concentration of B.

cenocepacia cultures. B. cenocepacia strains had overnight concentrations ranging from

approximately 1.2 x 1010 – 3.0 x 1010 and therefore the same dilution was sufficient for

each strain. It was found that the average overnight concentrations of strains 164 and

165 were 1.21 x 1010 CFU/mL and 2.32 x 1010 CFU/mL respectively. To calculate the MOI

needed for each infection assay, the concentration of bacteria needed from overnight

culture was 1 x 109 – 2 x 109 CFU/mL. It was found that a 1 in 10 dilution gave the most

accurate concentration range required when diluted to an OD600 of between 0.10 and

0.20 for both strains (Figure 3.4 and 3.5).

51


Overnight concentration of strain 164 cultures were an average of 1.21 x 1010



Overnight concentration of strain 165 cultures were an average of 2.32 x 1010


0.00E+00

2.00E+09

4.00E+09

6.00E+09

8.00E+09

1.00E+10

1.20E+10

1.40E+10

1.60E+10

0 0.05 0.1 0.15 0.2 0.25 0.3

CFU

/mL

OD600

1 in 20 1 in 10 3 in 20

0.00E+00

1.00E+09

2.00E+09

3.00E+09

4.00E+09

5.00E+09

6.00E+09

7.00E+09

0 0.1 0.2 0.3 0.4

CFU

/mL

OD600

1 in 20 1 in 10 3 in 20

52

Chapter Four


The assessment of K. pneumoniae and B. cenocepacia

adherence to RAW264.7 macrophage cells as a model for

inhibitor evaluation

53

4. The assessment of K. pneumoniae and B. cenocepacia adherence to RAW264.7

macrophage cells as a model for inhibitor evaluation

The developed assay method for testing the ability of K. pneumoniae and B.

cenocepacia to adhere to the cell surface of RAW264.7 macrophage cells is described in

section 2.2.3.4.1. Macrophages were pre-treated with cytochalasin-D for half an hour

prior to infection with bacteria at an MOI of 1:100-200 (Figure 4.1). Cytochalasin-D was

maintained at the same concentration throughout the experiment and a negative

control of macrophages only was present to confirm no contamination had occurred

throughout the experimental process. It was found that after washing to remove non-

adherent bacteria, K. pneumoniae and B. cenocepacia were adhering to the plastic of

the 24-well plate and thus a positive control of bacteria only with no macrophages was

implemented to compare the differences between the wells containing macrophages

plus bacteria (experimental wells) and the wells containing bacteria only (control wells).

Figure 4.1 Adherence assay model

Confluent monolayers of macrophages were pre-treated with 1 μg/mL cytochalasin-D

before infection with cytochalasin-D treated bacteria at an MOI of 1:100-200. Wells were

incubated for 1 hour to allow bacteria to adhere to the macrophages. Cells were then

washed, lysed and plated out on LB agar to determine viable bacterial counts.

54

4.1 K. pneumoniae adherence to RAW264.7 macrophage cells

Eight K. pneumoniae strains were examined on their adherence capabilities:

MGH78578, ST23.1, ST23.2, ST86, ST628, ST70, ST14 and ST770 (Figure 4.2). Strains

MGH78578, ST628, ST70, ST14 and ST770 showed little difference in viable bacterial

counts between the experimental wells and the control wells. This demonstrates that

these strains were able to bind to the macrophages, however, there was also strong

binding to the plastic of the 24-well plates. A difference between experimental wells and

control wells was observed in strains ST23.1, ST23.2 and ST86. This indicates that these

strains were also able to bind to the macrophages, but displayed binding to plastic at a

lesser extent, in comparison to the other strains mentioned above. A notable

observation was that strains ST23.1, ST23.2 and ST86 adhered to the macrophages at a

lower concentration than the other strains (< 1.6 x 105 CFU/mL). When a paired t-test

was conducted for statistical analysis between the experimental wells and the control

wells of strains ST23.1 and ST86, a significant difference was seen (P > 0.009 and P >

0.048 respectively). No significant difference was found for the other K. pneumoniae

strains.

55


macrophage cells

Macrophages were infected with K. pneumoniae for 1 hour at an MOI of

1:100. An average of three individual assays for each strain is graphed.

‘Bacteria + cells’ refers to the experimental wells and ‘bacteria only’ refers

to the control wells. The asterisks shown for certain strains indicate

significance. One asterisk signifies P ≤ 0.05 whereas two asterisks signify P ≤

0.01. The bars indicate SEM.

56

4.2 B. cenocepacia adherence to RAW264.7 macrophage cells

Three strains of B. cenocepacia were tested for their adherence capabilities to

RAW264.7 macrophage cells: 164, 165 and 167 (Figure 4.3). Experimentation for strains

164 and 167 were repeated three times for statistical significance. However, due to time

constraints, strain 165 was only experimentally repeated once and therefore no error

bars and statistical analysis could be calculated. A difference was observed between the

experimental wells and the control wells for all three strains. These results demonstrate

that the B. cenocepacia strains were able to specifically bind to the macrophages and

bind to the plastic of the 24-well plates, but to a lesser extent. The strains adhered to

the macrophages between an average of 3.5 x 105 and 5 x 105 CFU/mL but only adhered

to the plastic at an average of less than 2.5 x 105 CFU/mL. A paired t-test was conducted

for statistical analysis between the experimental wells and the control wells for strains

164 and 167 and it was found that there was no significant difference. However, large

error bars are seen which may explain why no significance was found.

57

Figure 4.3 Concentration of B. cenocepacia that adhered to RAW264.7

macrophage cells

Macrophages were infected with B. cenocepacia for 1 hour at an MOI of

1:200. An average of three individual assays for each strain is graphed.

‘Bacteria + cells’ refers to the experimental wells and ‘bacteria only’ refers

to the control wells. The hashtag (#) indicates that strain 165 was only

experimentally repeated once and therefore no statistical analysis or error

bars could be calculated. The bars indicate SEM in strains 164 and 167.

0

100000

200000

300000

400000

500000

600000

700000

164 165 167

CFU

/mL

Strains

Bacteria + cells Bacteria only

#

58

Chapter Five


The assessment of K. pneumoniae and B. cenocepacia

internalisation, survival and replication within RAW264.7


59

5. The assessment of K. pneumoniae and B. cenocepacia internalisation, survival and

replication within RAW264.7 macrophage cells as a model for inhibitor evaluation

The developed method for testing internalisation, survival and replication of K.

pneumoniae and B. cenocepacia is described in section 2.2.3.4. The macrophages were

infected with an MOI of 1:1000 for K. pneumoniae and 1:200 for B. cenocepacia (Figure

5.1). Two time points were examined, a 0 hour time point and a 24 hour time point post-

infection. A negative control of macrophages only was present to confirm that no

contamination had occurred throughout the experimental process and a positive control

of bacteria only was present to assure that all extracellular bacteria were killed during

the 1 mg/mL kanamycin kill step.

Figure 5.1 Internalisation, survival and replication assay model

Confluent monolayers of macrophages were infected with bacteria at an MOI of 1:1000 for

K. pneumoniae and 1:200 for B. cenocepacia. Wells were then incubated for 1 hour to allow

bacteria to internalise into the macrophages. The wells were washed and 1 mg/mL

kanamycin was added to kill all extracellular bacteria. Cells were then lysed and plated out

on LB agar to determine viable bacterial counts. Two time points were observed; 0 hour

and 24 hour post-infection.

60

5.1 K. pneumoniae internalisation, survival and replication within RAW264.7

macrophage cells

Seven strains of K. pneumoniae were tested for their ability to internalise, survive

and replicate in the macrophages: ST23.1, ST23.2, ST86, ST628, ST70, ST14 and ST770

(Figure 5.2). Two MOI ratios were tested, 1:100 and 1:1000. At the lower MOI no

bacterial counts were obtained at the 0 hour time point and the 24 hour time point for

all strains, suggesting that these strains either do not internalise into the macrophages

or that the concentration of K. pneumoniae added to the macrophages is too low for

successful internalisation. At the higher MOI of 1:1000, strains ST23.1, ST23.2, ST86,

ST70 and ST770 demonstrated no internalisation into the macrophages at the 0 hour

time point and the 24 hour time point (Figure 5.2). This demonstrates that these strains

were not able to internalise into macrophages even when a higher concentration of

bacteria was used.

Strains ST628 and ST14 were the only two strains that were able to internalise into

the macrophages at the 0 hour time point post-infection at an average of approximately

27 CFU/mL and 5 CFU/mL respectively (Figure 5.2). Strain ST628 was able to internalise

into the macrophages at the highest concentration in comparison to all other strains

however, was not able to survive and replicate during 24 hours in the macrophages as

no viable bacterial counts were obtained at this time point. Strain ST14 was the only

strain that was able to survive and replicate during 24 hours in the macrophages with a

5 fold increase in viable bacterial counts in comparison to the 0 hour time point. Even

though certain strains demonstrated internalisation, survival and replication in

macrophages, it was at very low concentrations of an average of less than 30 CFU/mL.

61

Figure 5.2 Concentration of K. pneumoniae that internalised, survived and



1:1000. An average of three individual assays for each strain is graphed. Two

time points were observed, a 0 hour time point and a 24 hour time point post-

infection. The bars indicate SEM.

0

5

10

15

20

25

30

35

40

ST23.1 ST23.2 ST86 ST628 ST70 ST14 ST770

CFU

/mL

Strains

0 hour 24 hour

62

5.2 B. cenocepacia internalisation, survival and replication within RAW264.7

macrophage cells

Three strains of B. cenocepacia were tested for their ability to internalise, survive

and replicate in macrophages: 164, 165 and 167. At an MOI of 1:200 all B. cenocepacia

strains were able to internalise into the macrophages at the 0 hour time point (Figure

6.2A). When comparing the same graph on a logarithmic scale and linear scale it is seen

that strain 165 was able to internalise at a much higher concentration (approximate

average of 4 x 105 CFU/mL) than the other two strains tested (an average of less than 5

x 104 CFU/mL in both strains 164 and 167) (Figure 6.2B). Furthermore, strain 165 was

the only strain that suggests replication in the macrophages during a 24 hour period,

with an approximate 1.25 fold increase in viable bacterial counts. In comparison, strains

164 and 167 declined in the number of viable bacteria during 24 hours in the

macrophages with an approximate 195 fold and 19 fold decrease respectively.

63

Figure 5.3 Concentration of B. cenocepacia that internalised, survived and


Macrophages were infected with B. cenocepacia for 1 hour at an MOI of 1:200.

An average of three individual assays for each strain is graphed. Two time points

were observed, a 0 hour time point and a 24 hour time point post-infection. ‘A’

is the results plotted on a logarithmic scale and ‘B’ is the results plotted on a

linear scale. The bars indicate SEM.

1

10

100

1000

10000

100000

1000000

164 165 167

CFU

/mL

Strains

0 hour 24 hour

A.

0

100000

200000

300000

400000

500000

600000

700000

164 165 167

CFU

/mL

Strains

0 hour 24 hour

B.

64

Chapter Six


The effect of Mip inhibitors on adherence and,

internalisation, survival and replication of K. pneumoniae

and B. cenocepacia in RAW264.7 macrophage cells

65

6. The effect of Mip inhibitors on adherence and internalisation, survival and

replication of K. pneumoniae and B. cenocepacia in RAW264.7 macrophage cells

The developed method for testing Mip inhibitors on adherence, internalisation,

survival and replication of K. pneumoniae and B. cenocepacia is described in section

2.2.3.5. Macrophages were infected with an MOI of 1:1000 for K. pneumoniae and 1:200

for B. cenocepacia (Figure 6.1). The Mip inhibitors have been developed to bear a

pipecoline moiety and resemble the rapamycin anchoring group but lack the macrocyclic

portion, thereby cancelling the immunosuppressive action.

The Mip inhibitors were dissolved in DMSO and thus a DMSO control was used to

verify that no differences seen were due to the effects of the DMSO in the experiment

rather than the Mip inhibitors. A negative control of macrophages only were present to

make sure no contamination had occurred during the experimental process. A positive

control of bacteria only was included for both adherence assays and internalisation,

survival and replication assays. In regards to the adherence assays this was to compare

the difference between bacteria only wells and the wells of the different variables (i.e.

Mip inhibitors and DMSO (control)). In regards to the internalisation, survival and

replication assays this was to assure that all extracellular were killed during the 1 mg/mL

kanamycin killing step. Macrophages plus bacteria control wells were also added to

make sure that bacterial counts that were being obtained were consistent with previous

adherence assay, and internalisation, survival and replication assay data.

66

Figure 6.1 Mip inhibitor testing models

Adherence assays (top pathway) and internalisation, survival and replication assays

(bottom pathway) were conducted as previously outlined with the same MOIs

however, to test the Mip inhibitors an added step was implemented as outlined in

the green box.

67

6.1. K. pneumoniae adherence to RAW264.7 macrophage cells with the Mip inhibitors

The results outlined in section 4.1 for K. pneumoniae adherence to macrophages

concluded that out of the eight strains tested, strains ST23.1 and ST86 showed a

significant difference between the experimental wells and the control wells at an MOI

of 1:100. Therefore, the Mip inhibitors were tested on these two strains to observe any

effects of K. pneumoniae adherence to the macrophages in the presence of inhibitor as

these strains were less able to bind to the plastic. Therefore, we can be confident that

the bacteria recovered are bacteria that had adhered to the macrophages specifically.

No difference in viable bacterial counts were observed between the DMSO control and

the Mip inhibitors 354 and 214 for strain ST86, suggesting that the Mip inhibitors do not

have an effect on adherence to RAW264.7 macrophage cells in this K. pneumoniae strain

(Figure 6.2). A decrease was observed in strain ST23.1 adherence with Mip inhibitor 354

by an approximate average of 1 x 105 CFU/mL in comparison to the DMSO control.

Surprisingly an increase in adherence was observed in strain ST23.1 adherence with Mip

inhibitor 214 in comparison to the DMSO control. Although there appeared to be a

difference between the Mip inhibitors and the DMSO control in the number of viable

bacterial counts obtained, no significant difference was found when a paired t-test was

conducted.

68




1:100 with Mip inhibitors 354 and 214 as well as DMSO (control). An average

of three individual assays for each strain is graphed. The bars indicate SEM.

0

20000

40000

60000

80000

100000

120000

140000

160000

ST23.1 ST86

CFU

/mL

Strains

DMSO I354 I214

69

6.2 B. cenocepacia adherence to RAW264.7 macrophage cells with the Mip inhibitors

The results outlined in section 4.2 for B. cenocepacia adherence to RAW264.7

macrophage cells found that no significant difference was observed between the

experimental wells and the control wells for all three strains tested. Due to the lack of

significance, limited time available and the limited Mip inhibitor available for testing, it

was decided that testing the inhibitors on B. cenocepacia adherence to the macrophages

will not go ahead.

6.3 K. pneumoniae internalisation, survival and replication within RAW264.7


The results outlined in section 5.1 concluded that K. pneumoniae strains ST628 and

ST14 were the only two strains out of the seven strains tested that internalised, survived

and replicated within the RAW264.7 macrophage cells at an MOI of 1:1000. Strain ST628

was internalised into the macrophages however was not able to survive and replicate

during 24 hours post-infection. Strain ST14 was the only strain to show internalisation,

survival and replication during 24 hours. Based on these results, strains ST628 and ST14

were chosen for Mip inhibitor testing. For strain ST628 only the 0 hour time point was

examined post-infection as no bacterial counts were observed at the 24 hour time point.

For strain ST14 however, inhibitors were tested at both the 0 hour time point and at the

24 hour time point.

When the Mip inhibitors 354 and 214 were tested in strain ST628 at the 0 hour time

point post-infection, statistical analysis found no significant difference (Figure 6.3). This

was perhaps due to the high variability of individual assays and thus, further repetition

of the experiments is required to increase statistical power. Therefore, to examine the

70

effects of the Mip inhibitors in the internalisation of this strain, trends were examined

across three individual replicates. Two of three assays conducted showed a decrease in

the number of viable bacteria obtained with the Mip inhibitors, in comparison to the

DMSO control. An approximate 40% decrease in viable bacterial counts was observed

with both inhibitors in experiment 1, and an 80% and 60% decrease in viable bacterial

counts was observed with inhibitor 354 and 214 respectively, in experiment 2. An

increase in the number of viable bacteria was observed with both inhibitors in

experiment 3 (Figure 6.3).

When testing the effect of the Mip inhibitors on strain ST14 it was found that the

inhibitors did not have a demonstrable effect on the internalisation, survival and

replication of this strain (Figure 6.4). Surprisingly, at the 0 hour time point an increase in

viable bacterial counts was observed for both inhibitors, particularly inhibitor 214, in

comparison to the DMSO control. Again at the 24 hour time point, an increase in the

number of viable bacteria that had survived and replicated in the macrophages was

observed with inhibitor 214. A slight decrease in the number of viable bacteria

(approximate average of > 2 CFU/mL) was suggested with the presence of inhibitor 354

at the 24 hour time point. However, statistical analysis of the data found no significance.

71

Figure 6.3 Concentration of K. pneumoniae strain ST628 that was

internalised into the RAW264.7 macrophage cells with the Mip inhibitors

Macrophages were infected with K. pneumoniae strain ST628 for 1 hour at

an MOI of 1:1000 with Mip inhibitors 354 and 214, as well as DMSO (control).

Results of three individual assays (experiments) are graphed to examine

trends in the data.

0

10

20

30

40

50

60

70

Experiment 1 Experiment 2 Experiment 3

CFU

/mL

DMSO I354 I214

72

Figure 6.4 Concentration of K. pneumoniae strain ST14 that internalised,

survived and replicated within RAW264.7 macrophage cells with the Mip

inhibitors

Macrophages were infected with K. pneumoniae strain ST14 for 1 hour at an

MOI of 1:1000 with Mip inhibitors 354 and 214, as well as DMSO (control). An

average of three individual assays for each strain is graphed. The bars indicate

SEM.

0

10

20

30

40

50

60

70

80

0 hour 24 hour

CFU

/mL

Time points

DMSO I354 I214

73

6.4 B. cenocepacia internalisation, survival and replication in RAW264.7 macrophage

cells with the Mip inhibitors

The results in section 6.2 concluded that the only B. cenocepacia strain which was

able to replicate in the RAW264.7 macrophage cells at an MOI of 1:200 during a 24 hour

period was strain 165. Strains 164 and 167 declined in viable bacterial counts during 24

hours in the macrophages. Therefore strain 165 was chosen for Mip inhibitor testing.

Mip inhibitors 354 and 214 were tested at both the 0 hour time point and at the 24 hour

time point post-infection to determine if there was a decrease in the number of viable

bacterial with inhibitor.

Statistical analysis of the data found that no significant difference was observed

when a paired t-test was conducted. This was due to the high variability of individual

assays and thus, further repetition of the experiments are required to increase statistical

power. Therefore, to examine the effects of the Mip inhibitors in the internalisation,

survival and replication of this strain, trends were examined across the three individual

experimental replicates (Figure 6.5). At the 0 hour time point, a decrease in viable

bacterial counts was observed with inhibitor 354 in comparison to the DMSO control in

two out of the three assays conducted. Experiment 3 in particular showed a 30%

decrease with inhibitor 354. Minor differences were observed with inhibitor 214 in

viable bacterial counts between individual assays at the 0 hour time point.

At the 24 hour time point, a decrease in viable bacterial counts were observed with

Mip inhibitor in two out of the three assays conducted. In experiment 1, there was an

approximate 30% decrease in viable bacterial counts with both inhibitors, and in

experiment 2 there was an approximate 20% and 30% decrease in viable bacterial

74

counts with inhibitor 354 and 214 respectively. Little change was observed in

experiment 3 with the inhibitors (Figure 6.5).

Figure 6.5 Concentration of B. cenocepacia strain 165 that internalised,

survived and replicated within RAW264.7 macrophage cells with the Mip

inhibitors

Macrophages were infected with B. cenocepacia strain 165 for 1 hour at an

MOI of 1:200 with Mip inhibitors 354 and 214, as well as DMSO (control).

Results of three individual assays (experiments) are graphed to examine

trends in the data.

75

Chapter Seven

Discussion

76

7. Discussion

K. pneumoniae and B. cenocepacia are important emerging pathogens and

contribute to the rise of MDR infections (Arnold et al. 2011; Zhou et al. 2007). K.

pneumoniae is capable of severe and possibly fatal infections, which include pneumonia,

urinary tract infections, liver abscesses and septicaemia (Prince et al. 1997; Lin et al.

2014; Pope et al. 2011). Secondary complications such as vision loss, necrotising fasciitis

and meningitis from metastatic spread occur (Siu et al. 2012). B. cenocepacia is most

common in individuals who have cystic fibrosis and chronic granulomatous disease due

to the immunodeficiency observed in these disease states, resulting in persistence of

infection (Speert 2002; Govan, Hughes & Vandamme 1996). Infections cause severe

pulmonary inflammation, pneumonia and septicaemia (Sajjan et al. 2008). ‘Cepacia

syndrome’ is a devastating characteristic of this pathogen causing rapid deterioration in

lung function and subsequent multi-organ failure (Blackburn et al. 2004).

Very few antibiotics remain universally effective for these pathogens. For example,

KPC strains are able to hydrolyse penicillins, all cephalosporins, monobactams,

carbapenems, and β-lactamase inhibitors (Papp-Wallace et al. 2010). B. cenocepacia is

naturally resistant to a wide range of antibiotics due to the many mechanisms of

antibiotic inhibition (Drevinek & Mahenthiralingam 2010). An increase in nosocomial

infections due to these pathogens, have resulted in prolonged morbidity and increased

mortality rates (Ramsay et al. 2013; Arnold et al. 2011). Therefore, the need for novel

inhibitors is evident. Bacteria exhibit many common virulence targets which can be

inhibited for potential broad-spectrum activity. For example, inhibiting quorum sensing

and cell division amongst others (O’Loughlin et al. 2013; Läppchen et al. 2005). A

virulence target which was first identified in L. pneumophila is the Mip protein

77

(Cianciotto et al. 1989). Other Mip-like proteins have been identified in B. pseudomallei,

C. trachomatis, N. gonorrhoeae and T. cruzi (Norville et al. 2011a; Lundemose, Kay &

Pearce 1993; Leuzzi et al. 2005; Moro et al. 1995).

Mip proteins have been identified as bacterial FKBPs which are involved in

intracellular virulence in a number of pathogens. FKBPs are highly conserved proteins

that bind to the immunosuppressive compounds FK506 and rapamycin, and display

peptidylprolyl cis/trans isomerase (PPIase) activity (Göthel & Marahiel 1999; Barik

2006). Due to their highly conserved nature, it is believed that putative Mip proteins are

present in K. pneumoniae and B. cenocepacia. Mip inhibitors have been developed to

mimic the rapamycin anchoring group but lack the immunosuppressive actions of the

compound. Therefore, they do not affect human FKBPs (Juli et al. 2011). When tested in

L. pneumophila and B. pseudomallei, a decrease in virulence was observed (Juli et al.

2011; Begley et al. 2014).

As Mip proteins have been identified in a range of bacteria, the inhibitors have been

proposed to have potential broad-spectrum activity. In order to assess this potential

broad-spectrum activity in vitro, a model must be developed. A few models are available

for in vitro testing which include biofilm models, 3D tissue-engineering models and cell

based models (Lourenço et al. 2014; Shepherd et al. 2009; Begley et al. 2014). These

models provide key aspects of infection and disease in a simplified manner for analytical

studies. Therefore, the aim of this study was to develop in vitro infection model methods

to test the potential broad-spectrum activity of the Mip inhibitors in K. pneumoniae and

B. cenocepacia.

78

7.1 Bioinformatic confirmation of putative Mip homologues in K. pneumonia and B.

cenocepacia

The first objective was to use bioinformatic tools to confirm the presence of

putative Mip homologues in K. pneumoniae and B. cenocepacia. This was done by

performing a BLAST search against the B. pseudomallei and L. pneumophila Mip

sequence. The putative K. pneumoniae Mip was identified to be 52% and 41%

homologous to the B. pseudomallei and L. pneumophila Mip respectively and the

putative B. cenocepacia Mip was identified to be 95% and 40% homologous to the B.

pseudomallei and L. pneumophila Mip respectively. These values are notable sequence

identities due to the many regions which were highly conserved between all four

pathogens (Figure 3.1).

Analysis of the L. pneumophila Mip identified that the protein is a surface expressed

lipoprotein that contains an N-terminal dimerisation and chaperone domain and a C-

terminal PPIase domain (Riboldi-Tunnicliffe et al. 2001). The active site of FK506, is a

cavity containing a number of hydrophobic residues that are highly conserved between

members of the FKBP family (Riboldi-Tunnicliffe et al. 2001). X-ray and NMR structural

investigation of the B. pseudomallei Mip observed that a number of amino acid side

chains displayed multiple conformations (Norville et al. 2011b). Most were distant from

the active site, however in the active site two amino acids in particular showed

significant flexibility; Aspartic acid44 and tyrosine89, outlined in the black boxes (Figure

3.1). It was found that the same flexibility was not observed in human FKBP12 (Szep et

al. 2009). These amino acids provide the only source of hydrogen-bond donors and

acceptors in the B. pseudomallei Mip active site and when these residues were mutated

in the L. pneumophila Mip, a significant reduction in enzyme activity was observed

79

(Wintermeyer et al. 1995). The unusual flexibility of these highly conserved side chains

suggest that they play a role in catalysis (Norville et al. 2011b).

As aspartic acid44 and tyrosine89 are highly conserved between all four pathogens,

and these amino acids have been identified as key residues in the active site of the B.

pseudomallei and L. pneumophila Mip, it is likely that the putative Mip proteins

identified in K. pneumoniae and B. cenocepacia are involved in virulence as well.

However, the proteins have not yet been studied in these pathogens. As the mutation

of these amino acids results in a significant reduction in enzyme function, the residues

are important drug binding domains. Therefore, based on the results of the sequence

alignment there is sufficient evidence that both K. pneumoniae and B. cenocepacia

contain Mip-like proteins. However, their function in these pathogens and whether they

are involved with virulence is still unknown.

7.2 The assessment of K. pneumoniae and B. cenocepacia adherence to RAW264.7


The second objective was to identify the magnitude at which K. pneumoniae and B.

cenocepacia adhere to RAW264.7 macrophage cells. This was achieved by developing

an adherence assay method which would be suitable for inhibitor evaluation of bacterial

adhesion to the macrophages, as described in section 2.2.3.4.1. The method developed

was simple, quantitative and required little equipment. The only technical challenge that

arose when conducting the adherence assays was that non-specific abiotic binding was

observed in the control wells for K. pneumoniae and B. cenocepacia. Therefore, even

though a confluent monolayer of macrophage cells was present at the bottom of each

experimental well, there was potential for non-specific abiotic binding to the sides of

the wells. An improvement would be to determine a way to measure specific binding.

80

For example, using microscopic enumeration of macrophage bound bacteria as a direct

measure rather than enumeration via plate counts. Despite this, the method described

in this study worked well with both K. pneumoniae and B. cenocepacia and

demonstrated that these pathogens were able to adhere specifically and non-

specifically.

7.2.1 K. pneumoniae adherence to RAW264.7 macrophage cells

K. pneumoniae strains MGH78578, ST628, ST70, ST14 and ST770 displayed specific

binding to macrophages but had strong non-specific binding to the plastic of the 24-well

plates (Figure 4.2). A difference was observed between the experimental wells and the

control wells of strains ST23.1, ST23.2 and ST86, suggesting that these strains were able

to adhere to the macrophages but were less able to adhere to the plastic. An interesting

observation of ST23.1, ST23.2 and ST86 was that they adhered to the macrophages at a

lower concentration than the other strains mentioned above.

Two major virulence factors which are linked to pathogenicity of K. pneumoniae are

the pili and capsular polysaccharides. Pili are found on the surface of the bacterium and

bind to host cells (Ofek & Beachey 1978; Venegas et al. 1995; Hornick et al. 1992;

Tarkkanen et al. 1997). They are also associated with biofilm formation on biotic and

abiotic surfaces (Schroll et al. 2010; Boddicker et al. 2006). Capsular polysaccharides

protect the bacterium from phagocytosis and serum killing (Podschun, Penner & Ullman

1992; Williams et al. 1983). Studies conducted on the relationship of capsule and

adherence have found that encapsulated K. pneumoniae strains impede the function of

type 1 pili and therefore decrease their ability to adhere to cells and agglutinate yeast

cells, which tests the mannose-binding phenotype of type 1 pili, because of the physical

interference of the capsule (Sahly et al. 2000; Schembri et al. 2005).

81

A unique feature of strains ST23.1, ST23.2 and ST86 that was not seen in the other

strains of this study was the mucoviscosity identified by the stretchability of the colonies

on agar plates when a bacterial loop was passed through. Even though the presence of

pili was not tested in this study, this occurrence could explain why a lower concentration

of bacteria was observed in strains ST23.1, ST23.2 and ST86. The over-expression of

capsule may physically hide the pili, lowering their ability to bind to the macrophages.

Pili are also partly responsible for non-specific binding to abiotic surfaces as well as

biofilm formation (Schroll et al. 2010; Di Martino et al. 2003). As the pili were most likely

hidden in these strains due to the thick capsule, this may also explain why a lower

concentration of viable bacteria were obtained in the control wells, in comparison to

the other strains tested (Figure 4.2).

The capsular type for strains ST23.1, ST23.2 and ST86 are K1, K1 and K2 respectively.

Epidemiological studies on the prevalence of various capsular types have found that K1

and K2 isolates are predominantly isolated from individuals in South East Asia, and cause

metastatic liver abscesses, pneumonia, urinary tract infections and bacteraemia (Liu,

Wang & Jiang 2013; Feizabadi, Raji & Delfani 2013; Ko et al. 2002). These capsular types

are also strongly associated with hypervirulent K. pneumoniae due to the mucoid

appearance and stretchability on agar plates, which is attributed to an increase in

capsule production by the acquisition of the rmpA gene located on a large virulence

plasmid (Cheng et al. 2010). As it has been demonstrated that capsule impedes the

function of pili due to a physical hindrance, and the literature suggests that strains of

thick mucoid capsules particularly of K1 and K2 capsular types are most associated with

MDR invasive infections, it is likely that other mechanisms that extend out of the thick

82

capsule may be involved in adherence to host cells as well as biofilm formation in these

strains.

In strains MGH78578, ST628, ST70, ST14 and ST770, little difference was observed

between the experimental wells and the control wells in the number of viable bacteria

obtained (Figure 4.2). In regards to these strains that were able to bind to the

macrophages but also strongly bind to the plastic, the capsule was not as mucoviscous

as they did not stretch from the agar plates when bacterial loops were passed through.

Based on this observation, it can be concluded that these strains are not mucoid

producing K. pneumoniae and therefore the capsule does not impede the function of pili

to the same extent as mucoid producing strains, allowing specific-binding to the

macrophages. The pili may also be exposed to allow abiotic adherence as well as biofilm

formation which may explain why a high number of viable bacteria were obtained in the

control wells. The formation of biofilms in the plastic may also contribute to the high

bacterial counts as they were still attached to the plastic even after washing.

7.2.2 B. cenocepacia adherence to RAW264.7 macrophage cells

B. cenocepacia strains 164, 165 and 167 were able to adhere to the macrophages

at an approximate average of 3.5 x 105 CFU/mL to 5.0 x 105 CFU/mL (Figure 4.3). Early

studies have identified that the adhesins of B. cenocepacia are important in adherence

and colonisation of the respiratory tract. In particular, the cable pili has been identified

as one of the main virulence factors of B. cenocepacia (Urban et al. 2005). Previous

analysis of these strains conducted by Adam Merritt from PathWest Laboratory

Medicine WA found that they do not contain the cblA gene needed for cable pili

formation. The absence of this gene did not affect these B. cenocepacia strains’ ability

to adhere to the macrophages and therefore other mechanisms are involved with

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adherence of B. cenocepacia to host cell. These results are consistent with a study that

created a cblA null mutant. No significant difference in adherence was observed in

mucin and epithelial cell attachment between the mutant strain and the wild-type

strain, suggesting that other mechanisms of adherence besides the cable pili are

involved (Tomich & Mohr 2003).

A study conducted by Mil-Homens and Fialho (2011) found that the highest number

and density of putative TAAs observed in the Bcc was in the B. cenocepacia genomovar.

It is unknown if all TAAs have unique functions or are redundant but this may explain

why B. cenocepacia has elevated pathogenicity in comparison to other Bcc species,

identified in the literature. The presence of TAAs along with other mechanisms have

been associated with biofilm formation, which may explain why bacterial counts were

observed in the control wells of the B. cenocepacia strains even after washing. It is

evident that B. cenocepacia has an array of mechanisms for adherence causing the

bacterium to be more pathogenic however, these mechanisms have not been fully

explored.

7.3 The assessment of K. pneumoniae and B. cenocepacia internalisation, survival and

replication within RAW264.7 macrophage cells as a model for inhibitor evaluation

The third objective was to identify the magnitude at which K. pneumoniae and B.

cenocepacia are internalised, survive and replicate in RAW264.7 macrophage cells after

24 hours post-infection. This was achieved by developing an internalisation, survival and

replication assay method which would be suitable for inhibitor evaluation in both

pathogens as described in section 2.2.3.4.2. For these assays two time points were

examined post-infection; a 0 hour time point and a 24 hour time point. Again, the assay

method was simple and quantitative and little equipment was required. No real

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improvements were needed for the developed method as sufficient results were

obtained for both pathogens, however, an alternative way of enumerating bacteria

would be to use fluorescent microscopy to count viable internalised bacteria. This would

be particularly useful for strains which are MDR, as was observed in the K. pneumoniae

strain MGH78578 of this study. This strain could not be killed with 1 mg/mL kanamycin

and therefore internalised bacteria could not be distinguished from external bacteria

based on plate counts.

7.3.1 K. pneumoniae internalisation, survival and replication within RAW264.7

macrophage cells

Results of the internalisation, survival and replication assays demonstrated that

two K. pneumoniae strains, ST628 and ST14, were internalised into the macrophages at

an MOI of 1:1000 after 1 hour of infection (Figure 5.2). Strain ST628 was internalised at

the 0 hour time point post-infection but was not able to survive and replicate during 24

hours in the macrophages. ST14 was the only strain that was internalised at the 0 hour

time point and replicated during 24 hours in the macrophages, with a 5 fold increase in

viable bacterial counts. All other strains were not internalised into the macrophages at

a high MOI after 1 hour of infection and thus seem to be extracellular pathogens, based

on the results of this study.

K. pneumoniae is known as an extracellular pathogen. However, it has been

demonstrated that these pathogens have the ability to internalise into epithelial cells

(Oelschlaeger & Tall 1997; Fumagalli et al. 1997; Hsu et al. 2015). Oelschlaeger and Tall

(1997) demonstrated that once attachment had occurred to the epithelial cell surface,

trigger mechanism(s) for invasion depended on microfilaments, microtubules and a

receptor which reflects receptor-mediated endocytosis. Local actin cytoskeleton

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rearrangement within the host leads to the formation of protrusions that engulf and

enclose the bacteria (Kochut & Dersch 2013). In addition to these mechanisms, Hsu et

al. (2015) found that hijacking host machinery Rho family GTPases and the

phosphatidylinositol 3-kinase/Akt signalling pathway is important in internalisation into

intestinal epithelium. Rho family GTPases control many processes including cell

proliferation and regulation of the actin cytoskeleton. Regulatory proteins of

phosphatidylinositol 3-kinase and Akt are also involved in many cell processes such as

the regulation of actin polymerisation and the promotion of microtubule stabilisation.

In order for K. pneumoniae to be internalised into host cells, it has been proposed that

the pathogen is capable of modulating phosphatidylinositol 3-kinase and Akt to control

both actin and microtubule dynamics during internalisation (Hsu et al. 2015).

When comparing the K2 capsular strains ST86 and ST14 it was observed that strain

ST86 produced mucoid colonies on agar plates however, strain ST14 did not appear to

have mucoid colonies on agar plates. Even though both strains are classified into the

same capsular type it seems that the overexpression of capsule is physically hiding the

adherence and possible internalisation mechanisms required for invasion in strain ST86

(Figure 5.2). As the capsule is not as prominent in strain ST14 the mechanisms required

for internalisation may be exposed resulting in internalisation of this K2 variant. Very

little is known about strain ST628 including the capsular type which limits the

conclusions that can be made however, it is evident that both ST628 and ST14 contain

mechanisms to promote internalisation into the macrophages which are not present in

the other strains of this study. Based on the results of these internalisation, survival and

replication assays in K. pneumoniae it is observed that internalisation is strain

86

dependent irrespective of capsular type and that many strains most likely exist as

extracellular pathogens.

7.3.2 B. cenocepacia internalisation, survival and replication within RAW264.7

macrophage cells

Martin and Mohr (2000) demonstrated that both a clinical isolate from the

epidemic ET12 lineage and an environmental isolate of B. cenocepacia are able to be

internalised into human macrophage and epithelial cells. When comparing the

intracellular growth of both these isolates over a 24 hour period, the environmental

isolate was not able to replicate over the 24 hour period however the epidemic clinical

strain increased by a log10 unit and then maintained this concentration between the 18

and 24 hour time points. The mechanisms associated with B. cenocepacia

internalisation, survival and replication have previously been investigated. It was found

that the bacterium’s flagella is important in initiation and contact of bacteria to host cell

and that a flagella formation gene knockout mutant had an impaired ability to invade

A549 epithelial cells (Tomich et al. 2002). Like L. pneumophila, B. cenocepacia is capable

of modulating phagosomal development as well as inhibiting the fusion of phagosome

to lysosomes, thereby allowing the bacteria to survive within the phagosome causing

persistent infections (Lamothe et al. 2007).

Two of the three clinical B. cenocepacia strains of this study were unable to

replicate during 24 hours in the macrophages. However, strain 165 was able to replicate

during a 24 hour period (Figure 5.3). This strain did not replicate a substantial amount

in comparison to the clinical isolate studied by Martin and Mohr (2000) however an

approximate average increase of 1 x 105 CFU/mL was observed. It is unknown at what

time point the strain replicated as only two time points were used in this study and

87

therefore it would be interesting to see if replication occurred early after post-infection

before becoming stagnant at the 24 hour time point, or if there was rapid replication

and then a slow decline near the 24 hour time point.

The results obtained from this study are consistent with past literature. Based on

the literature it seems strain 165 is able to evade phagosomal degradation by

modulating the maturation of phagosomes, survive within the phagosome, replicate and

promote dissemination. Strains 164 and 167 may not contain the required machinery to

evade phagosomal degradation and fusion to lysosomes however, as viable bacteria

were obtained after the 24 hour time point it seems that these strains are able to

prolong the process of phagosomal degradation. Thus, it was found that B. cenocepacia

can be internalised by macrophage cells, however the fate of these bacteria in their

ability to survive and replicate over a period of time is strain dependent.

7.4 The assessment of the effects of Mip inhibitors on adherence, and internalisation,

survival and replication of K. pneumoniae and B. cenocepacia within RAW264.7

macrophage cells

After identifying the presence of putative Mip proteins in K. pneumoniae and B.

cenocepacia as well as the magnitude at which these pathogens adhered, internalised,

survived and replicated in RAW264.7 macrophage cells, the final objective was to assess

the effects of Mip inhibitors 354 and 214 on infectivity of K. pneumoniae and B.

cenocepacia. This was achieved by adding an additional step to the adherence assay,

and internalisation, survival and replication assay methods as described in section

2.2.3.4.3. The extra incubation time with Mip inhibitor was not accounted for in the

assays without inhibitor and due to the high MOI that was used for K. pneumoniae

internalisation, survival and replication assays, this affected the experiments. The high

88

concentration of bacteria resulted in the media to change yellow prior to infection of

the macrophage cells; an indicator of low pH. The low pH could not sustain macrophage

cell survival and resulted in cell death. To overcome this, bacteria were centrifuged at

600 x g for 10 min after incubation with Mip inhibitors and then fresh media was added

to the same volume for subsequent macrophage cell infection. This slight change in the

method was only required for the assays with an MOI of 1:1000. No Mip studies in K.

pneumoniae and B. cenocepacia could be found when searching the literature and thus,

this is the first Mip study in these pathogens.

7.4.1 K. pneumoniae and B. cenocepacia adherence to RAW264.7 macrophage cells

with the Mip inhibitors

It is known that Mip proteins are involved with intracellular infection of a number

of pathogenic bacteria however, very little is known on whether these proteins are

involved with extracellular early stage infection prior to internalisation. In L.

pneumophila it was found that the first known extracellular function of Mip proteins

was its ability to bind to the extracellular matrix of lung epithelial cells and to collagen I-

VI via its C-terminal PPIase domain (Wagner et al. 2007). However, when looking at the

possibility of Mip proteins being involved with adherence, it was found that these

proteins were not involved with adherence of N. gonorrhoeae to macrophage cells and

B. pseudomallei to epithelial cells when compared to a knockout mutant (Leuzzi et al.

2005; Norville et al. 2011a).

Strains ST23.1 and ST86 were specifically chosen because not only were they K1

and K2 capsular type, which are most prevalent in K. pneumoniae infections, but a

significant difference was observed between the experimental wells and the control

wells. The significant difference implied that these strains were able to bind to the

89

macrophages but were less able to non-specifically bind to the plastic in comparison to

the other strains which displayed strong non-specific binding (Figure 4.2). Using strains

ST23.1 and ST86 assures that the bacteria obtained from inhibitor testing are most likely

bacteria that had adhered to the macrophages.

When testing the Mip inhibitors 354 and 214 in K. pneumoniae strains ST23.1 and

ST86 no significant difference was observed between the DMSO control and the

inhibitors for both strains, particularly in strain ST86 where very little difference was

observed (Figure 6.2). It seems that either Mip proteins are not present in this strain,

the Mip inhibitors are not binding efficiently to the putative protein’s active site or the

function of Mip is not involved in the adherence of bacteria to macrophages. In strain

ST23.1 a slight decrease in viable bacterial counts was observed with Mip inhibitor 354,

however, an increase in viable bacteria counts was observed with Mip inhibitor 214, in

comparison to the DMSO control. Despite this, these results were not significant and

large error bars were observed. Therefore, it is hard to draw conclusions on whether the

Mip inhibitors (particularly inhibitor 354) had an effect on adherence in this K.

pneumoniae strain and whether Mip proteins are involved with adherence to

macrophage cells.

As discussed previously, on further insight into the literature it was found that the

capsule of K. pneumoniae strains impede the function of pili due to the capsule

physically hiding the pili (Sahly et al. 2000; Schembri et al. 2005). As ST23.1 and ST86

were mucoid colony producing strains it is likely that the pili were hidden, decreasing

their ability to bind to the macrophages as was observed in the results (Figure 4.2).

Therefore it seems that the other non-mucoid producing strains would have been better

suited to test Mip inhibitors as the capsule would not impede function of pili to the same

90

extent as mucoid strains. Even though these strains show strong non-specific binding to

the plastic it would be interesting to see if Mip proteins have an effect on these strains,

as their ability to adhere to the macrophage cells is also higher (Figure 4.2).

The Mip inhibitors were not tested in B. cenocepacia due to the limited time

available as well as the limited Mip inhibitors available. The adherence assay results had

no significant difference between the experimental wells and the control wells and

therefore testing did not go ahead. Based on past literature it was observed that B.

pseudomallei Mip did not affect adherence to epithelial cells and as B. cenocepacia and

B. pseudomallei are in the same genus it is likely that the Mip proteins of each species

possess similar functions. If further time was available, it would be interesting to test

this premise as differences in bacterial counts are observed between the experimental

wells and the control wells of the adherence assays. Particularly when testing Mip

inhibitors in strain 165, which adhered at a higher concentration in comparison to strains

164 and 167. (Figure 4.3).

7.4.2 K. pneumoniae and B. cenocepacia internalisation, survival and replication

within RAW264.7 macrophage cells with the Mip inhibitors

Mip proteins have been demonstrated to be involved in intracellular infections of

L. pneumophila, N. gonorrhoeae, B. pseudomallei, C. trachomatis and T. cruzi (Cianciatto

et al. 1989; Leuzzi et al. 2005; Norville et al. 2011a; Lundemose, Kay & Pearce 1993;

Moro et al. 1995). In these pathogens, different functions of Mip have been identified.

For example, in L. pneumophila it appears Mip proteins are repressed directly after

internalisation but regain full activity after 24 hours of intracellular replication (Wieland

et al. 2002). In B. pseudomallei it is demonstrated that Mip proteins are important in

bacterial motility, protease production, and acid tolerance and in N. gonorrhoeae it was

91

demonstrated that the presence of Mip promoted intracellular survival (Norville et al.

2011a; Leuzzi et al. 2005).

K. pneumoniae strains ST628 and ST14 were tested with Mip inhibitor 354 and 214

as they were the only strains that were internalised into the macrophages (Figure 5.2).

ST14 was the only strain that was internalised, survived and replicated in the

macrophages during the 24 hours post-infection. Thus two time points were observed,

a 0 hour time and a 24 hour time point. Strain ST628 did not survive during 24 hours in

the macrophages and therefore only a 0 hour time point was observed. The results of

these assays demonstrated that in strain ST628 a trend in decreased viable bacterial

counts was observed with the presence of inhibitor, in comparison to the DMSO control.

Decreased viable bacterial counts ranged between 40 - 80% with inhibitor 354 and

between 40 - 60% with inhibitor 214, in two out of the three assays conducted (Figure

6.3). An increase in viable bacterial counts with inhibitor was observed in the third assay,

however, this is most likely due to introduced experimental errors. For example, maybe

the inhibitors were not fully dissolved and had formed crystals in solution, impacting on

the binding to Mip proteins. Based on these results it appears that Mip proteins play a

role in internalisation of this K. pneumoniae strain and early infection in macrophage

cells, however the exact mechanisms are not known.

In strain ST14 no significant effect was seen with the presence of Mip inhibitors at

both the 0 hour time point and the 24 hour time point post-infection (Figure 6.4). In fact,

an increase in viable bacteria was observed in comparison to the DMSO control for

inhibitor 354 at the 0 hour time point and inhibitor 214 at both time points. These results

suggest that Mip proteins may not be involved with intracellular virulence of this strain.

The increased viable bacterial counts observed with the inhibitors, in comparison to the

92

DMSO control is most likely attributed to high variation rather than the Mip inhibitors

promoting virulence in this strain.

B. cenocepacia strain 165 was tested with Mip inhibitors 354 and 214 as it was the

only strain that was capable of replicating during 24 hours in the macrophages. Both

strains 164 and 167 did not replicate during a 24 hour period and declined in the number

of viable bacteria (Figure 5.3). Thus, the 0 hour time point and 24 hour time point post-

infection were examined with inhibitors in strain 165. The results demonstrated that

inhibitor 354 had an effect at the 0 hour time point in two out of the three assays

conducted. However, very little difference was observed between inhibitor 214 and the

DMSO control (Figure 6.5). In B. cenocepacia flagella has been identified as an important

mechanism of the bacteria to internalise host cells (Tomich et al. 2002). In B.

pseudomallei the Mip knockout mutant was not able to produce flagella and had

significantly impaired motility (Norville et al. 2011a). Therefore, even though the

presence of flagella is unknown in strain 165, based on the results obtained it seems

that Mip proteins may affect flagella formation in B. cenocepacia. This is because a

decrease in viable bacterial counts were observed with inhibitor 354, in comparison to

the DMSO control (Figure 6.5).

At the 24 hour time point an approximate 20 - 30% decrease in viable bacterial

counts were observed in both inhibitors in two out of the three assays conducted.

(Figure 6.5). As mentioned above, it has been demonstrated that the L. pneumophila

Mip is supressed directly after internalisation but regains full activity after 24 hours

(Wieland et al. 2002). This conclusion may explain why the Mip inhibitors had an effect

at the 24 hour time point as the B. cenocepacia Mip may function in a similar manner to

93

the L. pneumophila Mip wherein full activity is seen after 24 hours of intracellular

replication.

7.5 Limitations and future work

As observed throughout all analysis, no statistically significant differences were

found despite apparent numerical differences between the controls and experimental

results, particularly when testing the Mip inhibitors in K. pneumoniae and B.

cenocepacia, due to the high variability of cell based experimentation. If more time was

available, these experiments should be repeated to increase the statistical power of the

study. Besides possible experimental error, a reason for high variation in numeric end

points observed in these experiments could be due to the specific concentration of

macrophages and bacteria used in each assay. Even though a concentration of 1 x 106

cells/mL and a bacterial dilution of 1 x 109 - 2 x 109 CFU/mL was aimed for, this count

was difficult to achieve exactly in each assay. Care was taken to ensure consistent

experimental conditions however changes in growth of the macrophages and bacteria

between individual assays will increase variability of the results.

Future work specific to this study could be to compare the effects of using different

MOIs, changing the incubation times for shorter or longer periods and having more time

points. A 1 hour incubation time was utilised for infection with macrophages and Mip

inhibitor-bacteria binding time, therefore, it would be interesting to see the impact this

has on infectivity of K. pneumoniae and B. cenocepacia with different incubation times.

Adding more time points for the internalisation, survival and replication assays will

provide key insight into the various stages of intracellular infection, with and without

Mip inhibitor. Using different strains of bacteria and different cell lines will also be

beneficial in acquiring a better understanding of the virulence of these pathogens,

94

particularly when comparing infection potential in epithelial cell lines and macrophage

cells lines. Testing the Mip inhibitors in a range of strains will increase knowledge of

strain variability, particularly in K. pneumoniae and cblA positive B. cenocepacia strains.

It has been identified by some studies that B. cenocepacia can be cytotoxic as was

observed in a cable pili positive isolate in epithelial cells (Cheung et al. 2007). Therefore,

conducting cytotoxicity assays alongside the plate count assays will provide comparisons

on pathogenicity of B. cenocepacia. Finally, as very little is known about the putative

Mip proteins of K. pneumoniae and B. cenocepacia, it is evident that an extension of this

study would be to characterise these proteins with molecular methods. For example,

creating a Mip knockout mutant of these pathogens and comparing virulence with the

wild-type strain.

7.6 Conclusion

In conclusion, this study has demonstrated that putative Mip proteins exist in K.

pneumoniae and B. cenocepacia. They are homologous to the B. pseudomallei and L.

pneumophila Mip, and contain highly conserved key amino acid residues of the active

site previously identified in B. pseudomallei. Based on the results, the adherence assay

model developed for these pathogens showed that all strains of K. pneumoniae and B.

cenocepacia were able to adhere to the macrophages, however, the concentration at

which they adhered varied between strains. The internalisation, survival and replication

assay model developed for K. pneumoniae and B. cenocepacia demonstrated that in K.

pneumoniae, not all strains were internalised into the macrophages, and only one strain

was able to replicate intracellularly at low concentrations. In comparison, all B.

cenocepacia strains were internalised into the macrophages, however, only one strain

was able to replicate intracellularly. When testing the Mip inhibitors on adherence of K.

95

pneumoniae, little effect was observed with the presence of the inhibitors. However,

when testing the Mip inhibitors on internalisation, survival and replication of K.

pneumoniae and B. cenocepacia, trends in the data suggest that the Mip inhibitors had

an effect on the intracellular virulence of these pathogens. Therefore from this

preliminary study, it seems that Mip inhibitors have potential broad-spectrum activity

in both K. pneumoniae and B. cenocepacia. In order to confirm this, characterisation of

the Mip proteins in both pathogens will need to be examined further.

96

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Appendix

Viable counts per mL = (number of colonies) x (dilution factor) x (volume factor)

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