Acinetobacter baumannii is an opportunistic nosocomial pathogen that causes ventilator-associated pneumoniae, bacteraemia, and wound and skin infections in immunocompromised individuals. A. baumannii can be multi-drug resistant and has become a concern for the global health care community, which must contain contamination and prescribe successful treatment for affected patients. The success of A. baumannii can be attributed to its plastic genome, which enables antimicrobial resistance, the ability to survive desiccation for extended periods, biofilm formation and capsule production to protect it from the human immune system. Capsule production by A. baumannii has been linked to antimicrobial resistance, biofilm formation, immune system evasion and desiccation persistence. Across the A. baumannii species, there are numerous capsule types that incorporate different sugars and configure them in different orientations. These capsule regions have been mapped and located across numerous strains, which suggests that the capsule locus is conserved. All capsule regions are flanked by the same genes: fkpA and lldP. To date, there has been no investigation of the possibility of the different capsule types affecting desiccation persistence, antimicrobial resistance, biofilm formation and immune evasion differently without background genetics influencing the results. The first aim of this study was to construct an operon assembly vector (OAV) system to investigate whether different capsule types will affect desiccation persistence, antimicrobial resistant, biofilm formation and immune evasion differently. OAV system construction involves three mains steps: (i) cloning an origin of replication specific to Acinetobacter spp., (ii) homologous recombination of the fkpA and lldP genes in yeast that will act as hook regions and (iii) reassembling the capsule biosynthesis operon (cps) locus from American type culture collection (ATCC) 17978 into the vector using homologous recombination in yeast. The first step of OAV system construction was achieved. The second aim of this study was to knockout the cps gene region in ATCC 17978 to create an isogenic mutant, ∆cps2, to enable the analysis of different capsule types using the OAV system. The isogenic mutant ∆cps2 was also characterised for resistance to desiccation, disinfectants and lysozyme to determine whether, without the capsule protecting the cell, the strain has reduced survival and therefore reduced persistence.

1. Effect of capsular polysaccharide in
Acinetobacter baumannii ATCC
17978 survival and construction of a
capsule operon recombination
system
An honours thesis submitted for the degree of
Bachelor of Science (Honours) at Flinders
University
Megan E. Cox
22 November 2018
College of Science and Engineering
Flinders University SA 5052, Australia

2. Page | i
Table of Contents
List of figures......................................................................................................................iv
List of tables.......................................................................................................................vi
Abstract ............................................................................................................................vii
Declaration ........................................................................................................................ix
Acknowledgements............................................................................................................ x
Chapter 1: Introduction...................................................................................................... 1
1.1 Acinetobacter baumannii.....................................................................................................1
1.1.1 Taxonomy .....................................................................................................................1
1.1.2 Epidemiology ................................................................................................................2
1.1.3 Pathology......................................................................................................................2
1.1.4 Virulence and persistence ............................................................................................3
1.2 Bacterial capsular polysaccharides.....................................................................................16
1.2.1 Capsule structure and serotypes................................................................................17
1.3 Acinetobacter baumannii capsular polysaccharides..........................................................21
1.3.1 Capsule production in Acinetobacter baumannii.......................................................23
1.3.2 Structure and serotypes of Acinetobacter baumannii ...............................................23
1.4 Scope of thesis....................................................................................................................26
Chapter 2: Materials and Methods................................................................................... 27
2.1 Bacterial strains used in this study.....................................................................................27
2.2 Bacterial growth media, buffers and solutions..................................................................27
2.3 Bacterial storage and growth conditions ...........................................................................33
2.4 Standard procedures..........................................................................................................33

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2.4.1 Purification of genomic DNA ......................................................................................33
2.4.2 Purification and isolation of plasmid DNA..................................................................34
2.4.3 Polymerase chain reaction .........................................................................................35
2.4.4 Agarose gel electrophoresis .......................................................................................42
2.4.5 Purification of polymerase chain reaction products ..................................................42
2.4.6 Digestion of DNA with restriction endonucleases......................................................43
2.4.7 Adenosine treatment of polymerase chain reaction products..................................43
2.4.8 Ligation of DNA...........................................................................................................44
2.4.9 DNA sequencing .........................................................................................................44
2.4.10 Transformation of Escherichia coli ...........................................................................44
2.4.11 Electroporation of Acinetobacter baumannii...........................................................46
2.5. Characterisation of Acinetobacter baumannii strains ......................................................48
2.5.1 Analysis of growth ......................................................................................................48
2.5.2 Lysozyme assay...........................................................................................................48
2.5.3 Desiccation assay........................................................................................................49
2.5.4 Sensitivity to disinfectants..........................................................................................49
2.5.5 Characterisation of capsule material..........................................................................50
Chapter 3: Results ............................................................................................................ 52
3.1 Introduction........................................................................................................................52
3.2 Construction of a cps deletion in Acinetobacter baumannii ATCC 17978 .........................55
3.3 Characterisation of ∆cps2...................................................................................................56
3.3.1 Verification of the absence capsule ...........................................................................58
3.3.2 Growth curve..............................................................................................................58
3.3.3 Lysozyme assay...........................................................................................................58

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3.3.4 Desiccation survival ....................................................................................................61
3.3.5 Resistance to antiseptics and disinfectants................................................................64
3.3.6 Colony morphology ....................................................................................................66
3.4 Cloning of the cps region....................................................................................................69
3.4.1 Development of the operon assembly vector using the operon assembly protocol.70
3.4.2 Re-design of the PW ori primer..................................................................................81
3.4.3 Design of the pW ori and tetracycline primer............................................................87
Chapter 4: Discussion....................................................................................................... 93
4.1 Characterisation of ∆cps2...................................................................................................93
4.2 The role of capsule in Acinetobacter baumannii survival..................................................94
4.3 Operon assembly vector system evaluation....................................................................102
4.4 Future directions for this research...................................................................................111
4.5 Conclusions.......................................................................................................................112
References ......................................................................................................................114
Appendices .....................................................................................................................129
Appendix 1: List of abbreviations...........................................................................................129
Appendix 2: Hyperladder 1.....................................................................................................132
Appendix 3: pGEM-T Easy Vector® map.................................................................................133
Appendix 4: pWH1266 map ...................................................................................................134
Appendix 5: pPR2274 map .....................................................................................................135
Appendix 6: Genes encoded on pPR2274 and pWH1277......................................................136

5. Page | iv
List of figures
Figure 1.1: Cluster comparison of capsular polysaccharide biosynthesis genes and the
corresponding capsule structure in representative Acinetobacter baumannii strains.........5
Figure 1.2: Types of surface glycoconjugates in Acinetobacter baumannii...............................8
Figure 1.3: Expected model of protein glycosylation during capsule synthesis from
Acinetobacter baumannii ATCC17978 taken from Lees-Miller et al. 2013.........................19
Figure 3.1: Cps gene region in ATCC 17978, ∆cps and ∆cps2 strains.......................................54
Figure 3.2: Homologous event in ATCC 17978 to create ∆cps2...............................................57
Figure 3.3: Alcian blue stain of extracted capsular polysaccharides from ATCC 17978 and
acapsular mutants, ∆cps2 and ∆cps in Acinetobacter baumannii ATCC 17978. .................59
Figure 3.4: Growth Curve of Acinetobacter baumannii strains WT, ∆cps and ∆cps2 at 37
°C in Mueller–Hinton broth under aerobic conditions........................................................60
Figure 3.5: The effect of lysozyme on Acinetobacter baumannii ATCC 17978 and
acapsular derivates..............................................................................................................62
Figure 3.6: Desiccation survival of capsular and acapsular strains in Acinetobacter
baumannii ATCC 17978. ......................................................................................................63
Figure 3.7: Resistance to chlorhexidine of Acinetobacter baumannii, ATCC 17978 and
acapsular derivates..............................................................................................................65
Figure 3.8: Resistance to benzalkonium chloride of Acinetobacter baumannii, ATCC
17978 and acapsular derivates............................................................................................67
Figure 3.9: Colony morphology of Acinetobacter baumannii ATCC 17978 and acapsular
derivates ∆cps and ∆cps2....................................................................................................68
Figure 3.10: The operon assembly protocol system developed by Liu et al. (2017). ..............72
Figure 3.11: Schematic representation of the cloning of pW ori and Gent into pPR2274. .....75

6. Page | v
Figure 3.12: Schematic representation of thymine to adenosine over-hang cloning of pW
ori and Gent into pGEM-T easy vector................................................................................78
Figure 3.13: Schematic representation of thymine to adenosine over-hang cloning of pW
ori into pGEM-T easy vector................................................................................................79
Figure 3.14: Schematic representation of thymine to adenosine over-hang cloning of
Gent into pGEM-T easy vector. ...........................................................................................80
Figure 3.15: Schematic representation of thymine to adenosine over-hang cloning of
nested product containing 2.5Kb pW ori and Gent cartridge into pGEM-T easy vector.
.............................................................................................................................................84
Figure 3.16: Schematic representation of restriction cloning of pW ori into pPR2274...........85
Figure 3.17: Schematic representation of restriction cloning of Tet into pPR2274. ...............86
Figure 3.18: Schematic representation of restriction cloning of 4.5Kb pW ori into
pPR2274...............................................................................................................................89
Figure 3.19: Agarose gel electrophoresis image of amplified 4.5 Kb pW ori from
transformed Escherichia coli DH5α with pPR2274..............................................................90
Figure 3.20: Agarose gel electrophoresis image of amplified 4.5Kb pW ori and amplified
250 bp of pPR2274 check from pW_2274...........................................................................91
Figure 4.1: Schematic representation of the completed operon assembly vector (OAV)
system................................................................................................................................104
Figure 4.2: Schematic representation of restriction cloning at the NsiI site with the
nested PCR product of mini-F and Tetracycline into pW_2274. .......................................109

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List of tables
Table 2.1: Bacterial strains used in this study..........................................................................28
Table 2.2: Growth media, buffers and solutions......................................................................29
Table 2.3: Primers used in this study .......................................................................................36
Table 2.4: Plasmids used in this study......................................................................................40

8. Page | vii
Abstract
Acinetobacter baumannii is an opportunistic nosocomial pathogen that causes ventilator-
associated pneumoniae, bacteraemia, and wound and skin infections in immunocompromised
individuals. A. baumannii can be multi-drug resistant and has become a concern for the global
health care community, which must contain contamination and prescribe successful
treatment for affected patients. The success of A. baumannii can be attributed to its plastic
genome, which enables antimicrobial resistance, the ability to survive desiccation for
extended periods, biofilm formation and capsule production to protect it from the human
immune system.
Capsule production by A. baumannii has been linked to antimicrobial resistance, biofilm
formation, immune system evasion and desiccation persistence. Across the A. baumannii
species, there are numerous capsule types that incorporate different sugars and configure
them in different orientations. These capsule regions have been mapped and located across
numerous strains, which suggests that the capsule locus is conserved. All capsule regions are
flanked by the same genes: fkpA and lldP. To date, there has been no investigation of the
possibility of the different capsule types affecting desiccation persistence, antimicrobial
resistance, biofilm formation and immune evasion differently without background genetics
influencing the results.
The first aim of this study was to construct an operon assembly vector (OAV) system to
investigate whether different capsule types will affect desiccation persistence, antimicrobial
resistant, biofilm formation and immune evasion differently. OAV system construction
involves three mains steps: (i) cloning an origin of replication specific to Acinetobacter spp.,

9. Page | viii
(ii) homologous recombination of the fkpA and lldP genes in yeast that will act as hook regions
and (iii) reassembling the capsule biosynthesis operon (cps) locus from American type culture
collection (ATCC) 17978 into the vector using homologous recombination in yeast. The first
step of OAV system construction was achieved.
The second aim of this study was to knockout the cps gene region in ATCC 17978 to create an
isogenic mutant, ∆cps2, to enable the analysis of different capsule types using the OAV
system. The isogenic mutant ∆cps2 was also characterised for resistance to desiccation,
disinfectants and lysozyme to determine whether, without the capsule protecting the cell, the
strain has reduced survival and therefore reduced persistence.

10. Page | ix
Declaration
I certify that this thesis does not incorporate without acknowledgment any material previously
submitted for a degree or diploma in any university; and that to the best of my knowledge and
belief it does not contain any material previously published or written by another person
except where due reference is made in the text.
Megan E. Cox

11. Page | x
Acknowledgements
I would like to thank and personally acknowledge Melissa Brown for giving me the opportunity
to carry out my honour’s year in her laboratory. I would also like to thank laboratory members
Jenny, Felise, Mohsen, Abol, Adele and Sylvia for their support, advice and encouragement
throughout this year.
Professional editor Dr Gillian Dite provided copyediting and document formatting services
according to standards D and E of the Australian Standards for Editing Practice and the
Guidelines for Editing Research Theses from the Institute of Professional Editors.
On a more personal note, I would like to thank my Ma, my Pa and my partner, Adnon. I would
also like to thank my twin sister, Erin, without whose statistical brilliance I would have been
lost. Without all of these people I would not have been able to submit this thesis. Their
support, knowledge and laughter were invaluable. To my honour’s year peers, Jess and
Shayne, without our coffee dates and chocolate-filled food comas, this year would have not
been as fun.

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Chapter 1: Introduction
1.1 Acinetobacter baumannii
1.1.1 Taxonomy
Acinetobacter baumannii is from the genus Acinetobacter (Giamarellou et al., 2008), which
comprises non-pigmented Gram-negative coccobacilli that can be oxidase-positive or negative
(Wong et al., 2017, Giamarellou et al., 2008). There is a large amount of diversity in the 50
species of Acinetobacter, which are generally non-pathogenic (avirulent) environmental
organisms (Wong et al., 2017). However, A. baumannii is an opportunistic pathogen in the
human host. Unlike other Acinetobacter spp., A. baumannii rarely colonises the skin of heathy
individuals (Wong et al., 2017, Al Atrouni et al., 2016).
A. baumannii is an opportunistic bacterial pathogen that is responsible for considerable
mortality and morbidity from nosocomial infections globally. Since the 1970s, the increased
incidence of nosocomial infections caused by multi-drug resistant (MDR) A. baumannii has
brought it to the forefront of clinical research (Dijkshoorn et al., 2007). Carbapenem-resistant
A. baumannii has been classified as priority 1 on the World Health Organization’s (WHO)
priority pathogen list for research and drug design for new antibiotics (WHO, 2017).
The increase in the incidence of infections caused by A. baumannii in intensive care units (ICU)
has been linked to increases in the use ofmechanical ventilation, catheterisation of the urinary
tract and bloodstream, and the use of antimicrobial treatments (Wong et al., 2017, Harding et
al., 2017). A. baumannii’s nosocomial success is attributed to its ability to survive long

13. Page | 2
desiccation periods, avoid the innate immune system of compromised individuals and rapidly
incorporate resistance genes in its genome to become multi-drug resistant, extremely drug
resistant and pan-drug resistant (Wong et al., 2017, Harding et al., 2017).
1.1.2 Epidemiology
A. baumannii is often acquired in hospital settings by immune-comprised individuals (Wong
et al., 2017, Tanguy et al., 2017, Dexter et al., 2015, Antunes et al., 2014, Eliopoulos et al.,
2008). In the nosocomial setting, A. baumannii can become widespread in its environmental
contamination (Tanguy et al., 2017) and has been reported to be found on curtains, medical
equipment, bed rails and cleaning equipment (Tanguy et al., 2017, Eliopoulos et al., 2008,
Zanetti et al., 2007).
A. baumannii infections are rarely reported outside hospitals but have been linked to natural
disasters, war zones and immune-impaired individuals such as alcoholics (Antunes et al., 2011,
Gaddy and Actis, 2009, Peleg et al., 2008, Eliopoulos et al., 2008, Dijkshoorn et al., 2007). All
A. baumannii infections are in immune-comprised individuals, which shows that this pathogen
is opportunistic rather than highly pathogenic (Antunes et al., 2011). A. baumannii infections
have led to increased hospital stays for patients and increased financial cost for treatment and
disinfection of hospital wards, equipment and personnel (Gandra et al., 2014).
1.1.3 Pathology
A. baumannii infections can be very severe due to the MDR status of the organism and
because patients who acquire the infection are at a higher risk of disease (Wong et al., 2017,

14. Page | 3
Tanguy et al., 2017, Dijkshoorn et al., 2007). The clinical manifestations of infection include
ventilator associated pneumonia, septic shock, tracheobronchitis, bacteraemia, skin and soft
tissue infections, biofilms at the site of surgery, and urinary tract infections (Martín-Aspas et
al., 2018, Wong et al., 2017, Dexter et al., 2015). Typically, clinically acquired A. baumannii
infections present as ventilator-associated pneumonia and community-acquired infections
present as pneumonia (Dikshit et al., 2017, Wieland et al., 2018). The mortality rate of A.
baumannii infections can range from 40% to 70% (Dikshit et al., 2017, Wieland et al., 2018).
The reduced ability of immune-comprised patients to fight against infection had led to
research investigating the virulence mechanisms that the clinical success of A. baumannii have
been attributed to (Harding et al., 2017).
1.1.4 Virulence and persistence
A. baumannii is a dynamic bacterium that can quickly adapt to changing environmental
conditions through its plastic genome (Martín-Aspas et al., 2018, Chin et al., 2018, Harding et
al., 2017). Because A. baumannii rapidly mutates when under stress or in adverse conditions,
this aids survival (Harding et al., 2017). In addition to A. baumannii’s plastic genome, the
essential virulence mechanisms that enable it to thrive in a health care environment and cause
disease are desiccation resistance, biofilm formation and motility, secretion systems, surface
glycoconjugates, and micronutrient acquisition systems (Martín-Aspas et al., 2018, Harding et
al., 2017, Wong et al., 2017).

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1.1.4.1 Plastic genome
A. baumannii is naturally competent, which means that it can readily take up foreign and self-
DNA and incorporate it into its genome (Imperi et al., 2011, Ramirez et al., 2010, de Vries and
Wackernagel, 2002). There are two mechanisms that A. baumannii uses to incorporate foreign
and self-DNA (Ramirez et al., 2010). Foreign DNA is incorporated through illegitimate
recombination (Hülter and Wackernagel, 2008), while self-DNA is incorporated through
homologous recombination (Domingues et al., 2012, Hülter and Wackernagel, 2008).
During the process of genetic sharing, horizontal gene transfer that involves gene acquisitions
and gene loss is part of genome evolution that allows A. baumannii to evolve quickly and
rapidly adapt to adverse environments (Domingues et al., 2012, Ramirez et al., 2010). The
genetic elements involved in horizontal gene transfer are gene cassettes, integrons,
transposases, insertion sequence elements and conjugative transposons (de Vries and
Wackernagel, 2002, Hülter and Wackernagel, 2008, Domingues et al., 2012). Newly
introduced DNA can be conjugated into different genes to change or adapt existing gene
functions. For example, in the A. baumannii capsule loci (KL) there are numerous insertion
sequence elements that result in adapted capsule structures (Wozniak and Waldor, 2010); see
Figure 1.1. These adapted structures may aid A. baumannii to improve its ability to persist and
survive (Giguère, 2015).

16. Page | 5
Figure 1.1: Cluster comparison of capsular polysaccharide biosynthesis genes and the
corresponding capsule structure in representative Acinetobacter baumannii strains.

17. Page | 6
(A) Capsule polysaccharide biosynthesis genes between fkpA and lldP were obtained from the
National Center for Biotechnology Information database and aligned using Easyfig 2.2.2. The
arrows indicate gene transcription direction, insertion sequence elements are indicated by
square boxes. Genes are colour matched based on homology to the putative function of gene
products, which can be found in the key. Sequence homology between capsule loci (KL)
regions are shown by a colour gradient. Genes are shown to scale. Genes that are involved in
synthesis of sugars that are of particular interest are Psep5Ac7RHb, 5-acetamido-3,5,7,9-
tetradeoxy-7-(3-hydroxybutanoylamino)-˪-glycero-˪-manno-non-2-ulosonic acid,
Psep5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-˪-glycero-˪-manno-non-2-ulosonic
(pseudaminic) acid, GlcNAc3NAcA4OAc, 2,3-diacetamido-2,3-dideoxy-α-ᴅ-glucuronic acid
with an additional O-acetyl group, ᴅ-GalpNAcA, N-acetyl-ᴅ-galactosaminuronic acid; ᴅ-
QuipNAc4NAc, 2,4-diacetamido-2,4,6-trideoxy-ᴅ-glucopyranose (N,N'-diacetyl-
bacillosamine); ᴅ-QuipNAc, N-acetyl-ᴅ-quinovosaminic acid, 8eLegp5Ac7Ac, 5,7-diacetamido-
3,5,7,9-tetradeoxy-˪-glycero-ᴅ-galacto-non-2-ulopyranosonic (di-N-acetyl-8-epilegionaminic)
acid; Aci5Ac7Ac, 5,7-di-N-acetylacinetaminic acid; ˪-FucpNAc, N-acetyl-˪-fucosaminic acid and
ᴅ-FucpNAc, N-acetyl-ᴅ-fucosaminic acid. Genbank accession numbers for the gene alignment
are KL37 KX712115.1 (23.4 Kb); KL93, CP021345.1 (30.3 Kb); KL6, KF130871.1 (25.5 Kb); KL2,
CP000863.1 (27.1 Kb); KL3, CP012004.1, (25.4 Kb); KL4, JN409449.3 (30.9 Kb); KL1, CP001172.1
(24.9 Kb) ); KL19, KU165787.1 (23.8 Kb); KL53, MH190222.1 (23.4 Kb region); KL49,
KT359616.1 (34.5 Kb region); KL13, MF522810.1 (38.2 Kb region) and KL12, JN107991.2 (38.5
Kb region). (B) Corresponding capsule structures to their KL gene regions shown in (A). K53,
K19 and K1 do not represent the percentage of O-acetylation of specific glycans in the
structural formation. Figure kindly supplied by Jennifer Singh and Felise Adams, College of
Science and Engineering, Flinders University.

18. Page | 7
1.1.4.2 Desiccation resistance
A. baumannii has adapted to be able to survive prolonged periods of desiccation, and this has
helped make it a nosocomial pathogen (Chin et al., 2018, Roca et al., 2012, Harding et al.,
2017). Desiccation resistance is the ability of a bacterium to remain viable under limited water
conditions (Harding et al., 2017). In Acinetobacter spp., desiccation resistance has been linked
to capsular polysaccharides (CPS) and the composition of the outer membrane of the
bacterium (Harding et al., 2017, Espinal et al., 2012, Boll et al., 2015). CPS promote survival
under desiccation by retaining water in the cell and contributing to the formation of biofilms
(Harding et al., 2017, Espinal et al., 2012). What is not known is if different capsule structures
change the bacterium’s ability to survive desiccation.
Another key component of A. baumannii’s outer membrane for desiccation resistance are the
acylated lipooligosaccharides (LOS) (Boll et al., 2015); see Figure 1.2 (6). LOS is a key glycan
structure that is anchored to the outer membrane through hepta-acylated lipid A (Hardings et
al., 2018); see Figure 1.2. LOS are important for a cell’s structural integrity and viability and
have been linked to drug and desiccation resistance (Boll et al., 2015, Hardings et al., 2018).
Strains with reduced acylated-LOS have weakened outer membrane structural integrity, which
results in diminished survival during desiccation (Harding et al., 2017, Boll et al., 2015). This
results in an increase of membrane fluidity and is likely to permit leakage of water and
hydrophilic nutrients into the environment, further starving the cell (Harding et al., 2017, Boll
et al., 2015).
A. baumannii overcomes the additional issue of re-hydration after desiccation, which is known
to cause DNA lesions (Harding et al., 2017, Aranda et al., 2011). These DNA lesions can occur

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Figure 1.2: Types of surface glycoconjugates in Acinetobacter baumannii.
The diagram shows the different types of surface glycoconjugates and export systems and
how they are oriented to the cell surface in A. baumannii. 1, shows lipid-linked oligosaccharide
(LLO) in with the outer membrane. 2, shows the different process that involve LLO. 3, shows
capsule production with liberated oligosaccharides from LLO. 4, shows how glycosylated
proteins are needed within biofilm formation. 5, shows that oligosaccharides are used to
glycated type IV pilins which involved in immune evasion. 6, shows the LOS structure within
the outer membrane. For more information refer to numbers 1 to 6 in text. This diagram was
taken from Harding et al. (2017).

20. Page | 9
in many forms, including oxidation, alkylation, base removal, crosslinking and strand breaks
(Potts, 1994). To eliminate some of the DNA damage resulting from rehydration after
desiccation, A. baumannii encodes and transcribes a protective protein RecA (Aranda et al.,
2011). RecA is an enzyme that is vital for homologous repair and recombination (Aranda et al.,
2011). Desiccation rehydration in A. baumannii has been hypothesised to contribute to MDR
profile due to an approximately 50-fold increase in the mutation frequency, which was
recorded as spontaneous rifampicin-resistant colonies were produced (Norton et al., 2013a).
The oxidative stress that occurs in the cell during desiccation is overcome by A. baumannii
upregulating proteins that detoxify reactive oxygen species (Harding et al., 2017, Gayoso et
al., 2013). All of these processes help A. baumannii to survive desiccation, allowing the cell to
persist in an unfavourable environment such as the nosocomial setting.
1.1.4.3 Biofilm formation and motility
Biofilms aid in bacterial survival through increased resistance to antimicrobial therapies,
environmental stresses, limited nutrient availability and desiccation (Gaddy and Actis, 2009,
Greene et al., 2016, Espinal et al., 2012). Within a biofilm, resistance to antimicrobial therapies
can increase to the order of one thousand times greater than that of a planktonic bacterium
(Gaddy and Actis, 2009). A biofilm is a slimy extracellular matrix where bacterial communities
are encased in an extracellular polymeric substance (Gaddy and Actis, 2009, Espinal et al.,
2012). The extracellular polymeric substance is made from carbohydrates, proteins, nucleic
acids and other macromolecules to create a barrier between the environment and the
bacterial community (Gaddy and Actis, 2009, Harding et al., 2017).

21. Page | 10
A. baumannii can form biofilms on both abiotic and biotic surfaces, which is one of the many
reasons why this pathogen can persist in a health care setting (Greene et al., 2016). A
planktonic A. baumannii cell produces pili to bind to either an abiotic or a biotic surface. This
is the first step in the adherence of cells to surfaces and imitates the microcolony formation
that precedes the development of biofilm structures (Gaddy and Actis, 2009). The pili are
produced from a polycistronic operon with six open-reading frames, 9o|
BABCDE in ATCC 17978 with additional loci that encode secretion functions that aid in pili
assembly and adhesion (Gaddy and Actis, 2009). Expression of this operon is tightly regulated
by a two-component regulatory system (Gaddy and Actis, 2009, Tomaras et al., 2008) that
comprises the sensor kinase bfmS and the response regulator bfmR (Tomaras et al., 2008).
Without BfmR, A. baumannii cannot produce pili and is unable to form a biofilm (Tomaras et
al., 2008). This is not seen when BfmS is removed, and suggests that the BfmR response
regulator talks to other sensing components in the cell (Tomaras et al., 2008). This implies that
different environmental stimuli could control biofilm formation (Gaddy and Actis, 2009,
Tomaras et al., 2008). For example, A. baumannii has been found to have increased ability to
form biofilms when there is resistance to broad-spectrum antibiotics and the presence of
metal cations (Gaddy and Actis, 2009). These factors contribute to A. baumannii forming
biofilms in the health care setting and help the bacterium to persist and remain viable (Greene
et al., 2016, Gaddy and Actis, 2009, Espinal et al., 2012).
1.1.4.4 Secretion systems
There are numerous protein secretion systems in A. baumannii (Weber et al., 2016). These
are extremely diverse in composition and function and are most often important mediators
of virulence (Johnson et al., 2016, Weber et al., 2016). The secretion systems that are well

22. Page | 11
known for their role in virulence and persistence are type II secretion systems,
autotransporters type V and type VI secretion systems (Weber et al., 2016, Bentancor et al.,
2012).
Type II secretion systems are found across the Acinetobacter species, suggesting that they are
essential for survival. Type II secretion systems are reported to aid in colonisation and survival
in different environments (Weber et al., 2016). LipA and LipH are lipases that break down lipids
and aid fatty acid metabolism (Johnson et al., 2016). This process provides a carbon surface
for the bacteria to digest and use as nutrient supply (Johnson et al., 2016, Weber et al., 2016).
CpaA is a metallopeptidase that degrades coagulated blood and is likely to aid in virulence
(Tilley et al., 2014). Without type II secretion systems, there is a reduction in bacterial load in
infections models (Weber et al., 2016).
Autotransporters are membrane-bound proteins where the C-terminal domain forms the
trimeric β-barrel, which allows the N-terminal passenger domain to transport macromolecules
to the bacterial cell surface (Bentancor et al., 2012). The autotransporter that has been
characterised in A. baumannii ATCC 17978 falls into the type V secretion system (Weber et al.,
2016, Bentancor et al., 2012). This transporter has been linked to important roles in biofilm
formation and binding to basal membrane or extracellular matrixes (Bentancor et al., 2012).
The ability to bind to basal membranes and extracellular matrixes is important for adhering to
host tissues; this is an important virulence factor when infecting hosts (Bentancor et al., 2012).
Autotransporters have been found to promote biofilm formation (Bentancor et al., 2012).

23. Page | 12
Type VI secretion systems have been linked to the production of antieukaryotic and
antibacterial toxins, which help the bacteria to out compete other bacteria and survive in the
host environment (Weber et al., 2016). The type VI secretion system injects effectors into
eukaryotic and bacterial cells. These actions bring the cell into a static phase, kill the cell or
stop the immune system from interacting with the cell (Carruthers et al., 2013). The type VI
secretion system is often lost because it requires a large amount of energy to be maintained
(Weber et al., 2016, Wright et al., 2014). Strains retaining type VI secretion systems are able
to out compete other bacteria in infection (Carruthers et al., 2013).
1.1.4.5 Micronutrient acquisition
All organisms need transition metals such as manganese, iron and zinc to live (Harding et al.,
2017). Because transition metals are so important for cellular processes, hosts have evolved
nutritional immunity that involves sequestering free metals such as manganese, iron and zinc
to stop infecting bacteria taking them (Harding et al., 2017). A key factor of the success of
A. baumannii as a nosocomial pathogen is its ability to scavenge scant nutrients such as
transitions metals in vivo (Harding et al., 2017, Wang et al., 2014). These metals become scant
during acute infection because the host decreases the surrounding pH level to acidic
conditions, thereby releasing the zinc chelating protein, calprotectin and other nutrient
immune responses (Harding et al., 2017, Shapiro and Wencewicz, 2015, Moore et al., 2014).
The key mechanism for scavenging free iron encompasses the use of siderophores, which are
high-affinity iron-chelating molecules (Harding et al., 2017). The siderophore that is most
frequently conserved in A. baumannii is the catechol-hydroxymate siderophore acinetobactin
(Shapiro and Wencewicz, 2015). The siderophore can be isomerised, depending on the

24. Page | 13
environmental pH, into two forms containing either oxazoline or isooazolidinone (Shapiro and
Wencewicz, 2015). Both these forms chelate free iron (Shapiro and Wencewicz, 2015). This
isomerisation of acinetobactin helps A. baumannii to chelate iron in acidic environments and
therefore overcome the host immune response (Harding et al., 2017). To combat calprotectin,
A. baumannii uses a high-affinity zinc acquisition system, ZnuABC (Hood et al., 2012). The
ZnuABC system is tightly regulated by a zinc uptake regulator protein (Zur) that is a
transcriptional repressor. The Zur binds on conserved DNA motifs upstream from zinc-
regulated genes to block expression. The Zur is released when the cell has depleted levels of
zinc, when calprotectin is present or under zinc depleted conditions. Subsequently, blocking
mediated by the Zur is relieved (Mortensen et al., 2014). These systems are all essential for
virulence and infection, and without these systems, the pathogenic A. baumannii are seriously
attenuated (Harding et al., 2017).
1.1.4.6 Efflux pumps
Efflux pumps are most commonly used either in removing and coping with hazardous
compounds such as antibiotics or toxins, or in removing harmful waste products from
metabolic processes (Du et al., 2018). Efflux pumps can also have roles in pathogenicity, cell-
to-cell communication and biofilm formation (Du et al., 2018). Efflux pumps are found across
the bacterial community and are seen in antimicrobial resistant phenotypes (Du et al., 2018).
Antimicrobial resistant phenotypes can evolve from overexpression, asymmetric increase
during division or from mutations occurring in genes that encode energy-dependent
transporters (Li et al., 2015). Efflux pumps can be divided into seven bacterial drug efflux pump
families: ATP-binding cassette (ABC), major facilitator superfamily (MFS), resistance-
nodulation-cell division (RND), multi-drug and toxin extrusion (MATE), small multi-drug

25. Page | 14
resistance (SMR), proteobacterial antimicrobial compound efflux (PACE) and antimetabolite
transporter (AbgT) families (Chitsaz and Brown, 2017).
The two most clinically relevant multi-drug resistant efflux pump families in A. baumannii are
RND and SMR (Du et al., 2018, Lin et al., 2017). One of these, the key efflux system AdeABC,
belongs to the RND family and produces resistance to cefotaxime, aminoglycosides,
erythromycin (Ery), chloramphenicol, fluoroquinolones, trimethoprim and tigecycline (Du et
al., 2018). The other key efflux system is AbeS, which belongs to the SMR family and produces
resistance to ciprofloxacin, chloramphenicol and Ery (Du et al., 2018). Regulators of efflux
pumps found in MDR A. baumannii are often found to contain mutants that allow for over
expression (Du et al., 2018). The over expression of efflux pumps contributes to a multi-drug
resistant phenotype and therefore aids in virulence and surviving the nosocomial setting (Du
et al., 2018).
1.1.4.7 Antibiotic resistance
Since the 1970s, MDR A. baumannii strains have become more prevalent among critically ill
patients and hospitals, to a concerning level (Dijkshoorn et al., 2007). Approximately 75% of
clinically isolated A. baumannii are MDR (Wieland et al., 2018), and pan-drug resistant strains
have large clinical impacts on treatment and patient outcomes (Wieland et al., 2018). These
dramatic adaptions to antibiotics occur through multiple cellular modifications, including
aminoglycoside modification, β-lactam hydrolysis, antibiotic target alterations, antibiotic
modification, active efflux pumps and changes to outer membrane proteins (Dijkshoorn et al.,
2007).

26. Page | 15
Resistance to carbapenems and broad spectrum β-lactams are particularly concerning
because these have been the most important antibiotics in the treatment of infections with
A. baumannii (Dijkshoorn et al., 2007). One of the reasons why A. baumannii has accumulated
so many antibiotic resistance mechanisms is its natural ability to incorporate foreign and self-
DNA (Imperi et al., 2011, Dijkshoorn et al., 2007); see Section 1.1.4.1.
1.1.4.8 Surface glycoconjugates
Surface glycoconjugates are bacterial carbohydrates (glycans) that are associated with the
outer membrane of the bacterium(Harding et al., 2017); see Figure 1.2. Surface
glycoconjugates create a barrier between the environment and the bacterial cell (Harding et
al., 2017, Hug and Feldman, 2011). These barriers are key structures that form the first line of
defence against environmental stressors, immune evasion or regulation, and virulence
(Harding et al., 2017, Wang-Lin et al., 2017). Standard bacterial glycoconjugates consist of
glycosylated proteins, lipopolysaccharides and peptidoglycan (Harding et al., 2017); see Figure
1.2. These all contribute to A. baumannii virulence (Harding et al., 2017).
Glycan synthesis starts at the inner membrane of A. baumannii where dedicated
glycotransferases transfer sugars onto a phosphorylated lipid that creates a lipid linked
oligosaccharide (Scott et al., 2014, Iwashkiw et al., 2012); see Figure 1.2 (1) LLO. The lipid link
oligosaccharide is flipped to the periplasm (Scott et al., 2014, Harding et al., 2017); see Figure
1.2 (2). This can then lead to one of three processes to produce capsule, glycosylated proteins
or glycosylated type IV pilins using oligosccharyltransferases (Harding et al., 2017); see Figure
1.2 (3, 4 and 5, respectively). A. baumannii glycosylated proteins contribute to virulence by
aiding biofilm formation and maintenance (Iwashkiw et al., 2012). The capsule produced by

27. Page | 16
A. baumannii protects it from host complement-mediated killing (Russo et al., 2010).
Glycosylated type IV pilins in A. baumannii have been linked to protecting bacterial proteins
from antibody recognition (Harding et al., 2017, Piepenbrink et al., 2016). Finally, LOS in the
core glycan that are anchored to lipid A with no O antigen directly contribute to drug and
desiccation resistance (Boll et al., 2015); see Figure 1.2 (6).
1.2 Bacterial capsular polysaccharides
Several Gram-negative and Gram-positive bacteria produce a layer of polysaccharides, called
the capsule, which is associated with the outer membrane and encapsulates the bacteria
(Willis and Whitfield, 2013); see Figure 1.2 (3). The polymers that comprise the capsule are of
high molecular weight and contain oligosaccharide units that undergo polymerisation to form
a long chain (Kenyon et al., 2014). These repeat units are joined by specific linkages that are
catalysed by glycosyltransferases (Kenyon et al., 2014). Sugars and glycotransferases are used
to create a variety of capsule structures (Mostowy and Holt, 2018). This occurs through
different chemical bonds being used to join the oligosaccharides to create a single sugar
structure that is repeatedly joined to create the capsule (Mostowy and Holt, 2018). This
creates diversity in CPS and therefore results in different serotypes (Mostowy and Holt, 2018).
The polymers act as a glycan shield that protects the cell from external stressors (Chin et al.,
2018, Willis and Whitfield, 2013). The sugar-based matrix is generally hydrophilic in nature,
which aids in retaining water in the cell (Chin et al., 2018, Harding et al., 2017). The capsule is
not crucial for survival but it is used in surviving desiccation, providing protective immunity
and having an essential role in virulence, a feature which is shared across bacterial species

28. Page | 17
(Harding et al., 2017, Yother, 2011). As well as being important for colonisation and causing
diseases (Mostowy and Holt, 2018), one of the key points for its role in pathogenesis in
humans is that it protects the bacterium from the complement system, antibodies and
engulfment from macrophages (Mostowy and Holt, 2018). An example where a capsule is
important for pathogenicity in the human host is Streptococcus pneumoniae (Yother, 2011).
1.2.1 Capsule structure and serotypes
The polysaccharide chains that comprise the capsule are produced in different biosynthetic
ways (Willis and Whitfield, 2013, Raetz and Whitfield, 2002). The three primary biosynthetic
pathways seen across the bacteria kingdom are the Wzy-dependent, synthase-dependent and
ABC-transporter dependent pathways, of which the two most common are the Wzy-
dependent and ABC-transporter dependent pathways (Raetz and Whitfield, 2002). Only the
Wzy-dependent pathway is found in both Gram-negative and Gram-positive bacteria (Raetz
and Whitfield, 2002). The synthase dependent pathway is less common in bacteria (Raetz and
Whitfield, 2002, Yother, 2011). The biosynthetic pathways involved in the three primary
capsule synthesis mechanisms are as diverse and different as the genes that encode for the
sugars that comprise the polysaccharide chain (Yother, 2011).
The ABC-transporter dependent pathway is limited to producing linear O-polysaccharides
(Raetz and Whitfield, 2002). The undecaprenyl phosphate lipid (und-PP) carrier-linked
polysaccharide chain grows from the addition of glycosyl residues to the non-reducing
terminal (Raetz and Whitfield, 2002). Und-PP, undecaprenyl pyrophosphate lipid carrier is
used as a scaffold for the growing polysaccharide chain (Whitfield, 2006). The polymerase

29. Page | 18
enzymes involved in attaching new carbohydrates to the chain are not specific to this system.
The polymer chain is constructed on the inner face of the cytoplasmic membrane where
export and ligation occurs through the ABC transporter without involvement from the Wz
complex (Raetz and Whitfield, 2002); see Figure 1.3. The Wz complex involves the proteins
wza, wzy, wzb and wzc, which are shown in red in Figure 1.3 (Raetz and Whitfield, 2002, Lees-
Miller et al., 2013).
The Wzy-dependent pathway involves single undecaprenyl-linked O-repeating units being
transferred across the inner membrane to the periplasmic space by the Wz complex (Raetz
and Whitfield, 2002). The Wzy complex is attached to the Wz complex and produces a putative
polymerase that uses the single undecaprenyl-linked O-repeating unit as substrates for chain
extension in the periplasm (Raetz and Whitfield, 2002). At the reducing terminus, chain
extension occurs and the nascent chain is transferred from the undecaprenyl linker to the non-
reducing terminus of another undecaprenyl-linked sub-unit (Raetz and Whitfield, 2002). The
chain length and how much polymerisation occurs is determined by the Wzz complex, which
is attached to the Wz complex (Raetz and Whitfield, 2002). Once the nascent polymer chain
has been ligated to a lipid-A core, translocation occurs to the outer membrane (Raetz and
Whitfield, 2002).
The synthase-dependent pathway involves the glycotransferases WbbE, WecA and WbbF
(Raetz and Whitfield, 2002). WecA creates the primer, the start of the polymer chain, and then
WbbE then joins an adaptor to the chain, leading to chain extension by WbbF (Raetz and
Whitfield, 2002). WbbF is a dual linkage processive glycotransferase that is involved in
exporting the undecaprenyl-linked intermediates into the periplasm

30. Page | 19
Figure 1.3: Expected model of protein glycosylation during capsule synthesis from
Acinetobacter baumannii ATCC17978 taken from Lees-Miller et al. 2013.

31. Page | 20
(Raetz and Whitfield, 2002). Chain extension occurs by the WbbF glycotransferase, which links
repeat units (Raetz and Whitfield, 2002). The nascent polymer chain is then ligated to a lipid
A core before being translocated to the outer membrane (Raetz and Whitfield, 2002). The
polymer produced from this biosynthetic pathway is simpler than that from the Wzy
dependent pathway because it normally contains only one or two sugars (Yother, 2011).
However, the polymer can be branched or linear, and is therefore more complex than that
from the ABC-transport dependent pathway (Yother, 2011). Branching is determined through
the linkages formed during polymerisation of the sugars (Yother, 2011).
How the different sugars and branches are incorporated into the polymers determines the
capsule type, which is known as a serotype (Yother, 2011). Different serotypes are known to
invoke different immune responses in the host (Jochems et al., 2017). This then affects the
distribution of bacteria with those specific serotypes. For example, S. pneumoniae serotype
6B has declined prevalence since the release of the conjugate vaccine PCV7 (Jochems et al.,
2017, Balsells et al., 2017).
Bacteria have evolved the ability to avoid being recognised and phagocytised by the immune
system by swapping capsule regions through horizontal gene transfer (Chin et al., 2018). The
mechanism that allows capsule swap mutants to occur is homologous recombination (Yother,
2011), which can occur through anchoring highly conserved genes that flank either side of the
capsule region (Willis and Whitfield, 2013, Yother, 2011). This allows the genetic material that
lies between the two flanking genes to be swapped with capsule genes that have been taken
in by the cell through horizontal gene transfer (Yother, 2011, Mostowy and Holt, 2018). This

32. Page | 21
can create a different serotype of the bacterial strain and can provide immune evasion (Coffey
et al., 1998, Yother, 2011).
The constant evolution of capsule regions can affect the development of vaccines that target
the whole bacterial species and not just a specific serotype (Balsells et al., 2017). This is seen
in vaccination for S. pneumoniae, which has vaccines that target the most common capsule
types but fail to target the whole species (Balsells et al., 2017). Vaccination has affected
serotype prevalence rates for S. pneumoniae, and therefore affects the serotypes that are
important for clinical disease in the wider community (Chen et al., 2018). This shows how
important the capsule is to bacterial species as a virulence factor and enabling persistence in
a clinical setting (Yother, 2011).
1.3 Acinetobacter baumannii capsular polysaccharides
To date, capsules in A. baumannii strains have only been investigated to determine the effect
of bacterial survival in different environmental stressors (Martín-Aspas et al., 2018, Chin et al.,
2018, Harding et al., 2017). The presence of a capsule has been shown to be a major virulence
factor in A. baumannii, as with most bacterial strains that have capsules (Lees-Miller et al.,
2013, Chin et al., 2018). The presence of a capsule is linked to the pathogenicity of
A. baumannii. Without a capsule the bacteria cannot survive in human serum, grow in human
ascites fluid and remain virulent (Chin et al., 2018, Russo et al., 2013).
Encapsulated A. baumannii strains have been found to have higher rates of bacterial loading
in mouse infection models and have higher colony forming units per millilitre (CFU/mL) in the

33. Page | 22
lungs, spleen and liver (Chin et al., 2018). Capsules help A. baumannii to persist the clinical
environment by retaining water to keep the cell viable (Harding et al., 2017, Espinal et al.,
2012) and are important in initiating biofilm formation, which helps the bacteria persist in the
clinical environment, including infection in patients (Harding et al., 2017, Greene et al., 2016).
In ATCC 17978 (K3), up-regulation of capsule production increases serum resistance and
virulence in the mouse model (Geisinger and Isberg, 2015). This is also seen in the phase
variant strain A. baumannii AB5075, which can transition between opaque and translucent
colonies. The translucent colonies have reduced capsule production and lower bacterial load
compared with the opaque colony, which produces two-fold thicker capsule and has a 10,000-
fold higher bacteria load in the lungs (Chin et al., 2018). This further identifies how important
capsule is in the pathogenicity of A. baumannii in a mammalian model.
Currently, there are no licensed vaccines or non-antibiotic treatments for A. baumannii
infection, but recently there has been increased interest in their development due to the WHO
classification of A. baumannii as priority 1 critical organisms for the development of new
antimicrobials (WHO, 2017). Recent studies have shown that the efficacy of passive
immunisation using a CPS-specific antibody and conjugate vaccines using a protein carrier to
exposed purified CPS elicits a protective immune response (Yang et al., 2018, Russo et al.,
2013). Further understanding of the role of capsules in multiple strains of A. baumannii is
essential for identifying the best targets for new vaccines and for establishing whether the
development of new antimicrobials that target capsule synthesis is even possible.

34. Page | 23
1.3.1 Capsule production in Acinetobacter baumannii
In A. baumannii, capsule construction and export occur through the Wzy-dependent pathway
(Russo et al., 2010, Kenyon et al., 2015a). The oligosaccharide sub-units that form the capsule
are termed K units (Russo et al., 2010). K units typically comprised between four to six sugars
and are built on the Und-PP lipid carrier (Whitfield, 2006); see Figure 1.1 (B). The Wzy-
dependent pathway produces the linked oligosaccharide chains, and the Wz complex exports
the capsule polymers to the outer membrane (Figure 1.3).
Environmental conditions such as temperature, metabolite availability, ion availability and
osmotic pressure can influence CPS production, as can sub-inhibitory levels of antimicrobials
(Geisinger and Isberg, 2015). A. baumannii regulates capsule production through two
identified transduction systems: BfmRS and OmpR-EnvZ (Geisinger and Isberg, 2015, Tipton
and Rather, 2017). Both BfmRS and OmpR-EnvZ play regulatory roles in envelope biogenesis
(Geisinger and Isberg, 2015). In ATCC 17978, capsule biosynthesis operon (cps) expression can
increase when the bacteria are subjected to antibiotic pressure in a BfmRS-dependent manner
(Geisinger and Isberg, 2015). CPS production and phase variation in A. baumannii AB5075 is
highly regulated by the OmpR-EnvZ system (Tipton and Rather, 2017). Phase variation from
translucent to opaque in AB5075 is from a two-fold increase in capsule thickness, which results
in higher pathogenicity (Chin et al., 2018, Tipton and Rather, 2017).
1.3.2 Structure and serotypes of Acinetobacter baumannii
The genes that encode capsule production are located in the KL locus in A. baumannii (Kenyon
et al., 2014, Kenyon and Hall, 2013). The KL locus encodes for capsule production, capsule

35. Page | 24
export and the enzymes that synthesise the linkage of oligosaccharide units (Kenyon et al.,
2014, Kenyon and Hall, 2013). Over 100 different KL loci have been identified in A. baumannii
(Shashkov et al., 2017). These KL regions typically range from 20 to 35 Kb in size (Shashkov et
al., 2017)and lie between the fkpA and lldP genes, which are highly conserved throughout the
species (Kenyon et al., 2014, Kenyon and Hall, 2013); see Figure 1.1 (A). A. baumannii strains
that possess the KL19 and KL39 regions have a Wzy polymerase encoded elsewhere on the
chromosome (Kenyon et al. 2016). A. baumannii KL regions have a similar arrangement with
highly conserved CPS transportation genes shown in orange and containing the Wz complex
(Kenyon and Hall, 2013); see Figure 1.1 (A) and Figure 1.3. Highly variable regions of synthesis
and transferase genes are required for complex sugars to be synthesised (green), which leads
into conserved simple sugars (blue) for CPS production (Figure 1.1 (A)). Repeat unit processing
is undertaken by wzx and wzy genes, which are highly variable between strains (light blue,
Figure 1.1 (A)).
The highly conserved structure of the genes fkpA and lldP on either side of the KL loci in A.
baumannii show how adapted this region is to be modified and exchanged between different
bacteria (Mostowy and Holt, 2018, Kenyon and Hall, 2013); see Figure 1.1. With the KL gene
clusters varying to create both simple and complex K unit structures, A. baumannii can retain
redundant duplicated genes (itr genes in KL8) and insertion sequence elements to contribute
to the diversity seen in these KL regions (Shashkov et al., 2016, Kenyon and Hall, 2013, Lees-
Miller et al., 2013). KL regions can reveal a lot about the corresponding K unit structures, but
they still need to undergo biochemical testing and chemical analysis to determine their exact
structures and the linkages between sugars (Kenyon et al., 2014, Kenyon and Hall, 2013,
Mostowy and Holt, 2018).

36. Page | 25
KL region diversity translates into the diversity seen in K unit structures in A. baumannii
(Kenyon and Hall, 2013, Kenyon et al., 2015a, Kenyon et al., 2014). Over forty K unit structures
have been mapped using nuclear magnetic resonance (NMR) spectroscopy. The sugar
composition of K units can vary and can include common Und-PP linked sugars such as glucose,
glucuronic acid and galactose or rare sugars like non-2-ulosonic acids (Shashkov et al., 2017).
The K units structures differ in length from five to six mono-saccharides (K37) to only two
residues (K53) (Figure 1.1 (B)). This also occurs with the linkages between K units, which can
be linear (K1) or have numerous side branches (K93), as shown in Figure 1.1 (B) (Kenyon et al.,
2017). Other factors that contribute to the diversity seen in the K units are the level of O-
acetylation patterns and the specific glycosidic bonds of the oligosaccharides (Kenyon et al.,
2017). The differences between K units can be prominent through the addition of rare sugars:
pseudominic (K2/6), acinetaminic acid (K12/K13) or legionaminic (K49) derivatives (Figure 1.1
(B)). The K unit structures can also have minor differences arising from the linkage of two
glycans that use different Wzy polymerases (K12, K13), as shown in Figure 1.1 (B) (Kenyon et
al., 2014). Acinetaminic acid derivatives have only been identified in A. baumannii, not
elsewhere in nature (Kenyon et al., 2017).
Previous research has not addressed whether different capsule types interact differently with
desiccation, biofilm formation, host survival and antibiotic resistance. This information is vital
to understanding the role of diverse capsule types in the pathogenicity of A. baumannii. More
information on role of CPS in pathogenesis is an important step towards the ultimate aim of
improving morbidity and mortality rates in infected patients (Chin et al., 2018, Kenyon et al.,
2017, Geisinger and Isberg, 2015).

37. Page | 26
1.4 Scope of thesis
The aims of this study were twofold. The first aim was to address the gap in our knowledge of
the role of capsules in A. baumannii pathogenicity by using an operon assembly protocol (OAP)
developed by Liu et al. (2017), to create an operon assembly vector (OAV) specialised for
A. baumannii. This OAV system can be used to produce different capsule types using a single
plasmid, which involves use of the double strain repair pathway in yeast.
The second aim of this study was to create a capsule knockout mutant, ∆cps2, that has the KL
locus from fkpA to lldP replaced with an Ery cartridge. The knockout mutant was created in A.
baumannii ATCC 17978 because this strain has fewer resistant cartridges than other A.
baumannii strains. The resulting capsule knockout mutant, ∆cps2, was the isogenic model for
the OAV system expression. The ∆cps2 mutant was be characterised to understand the direct
role that the capsule plays in resistance to desiccation, disinfectants and lysozyme. This
included identifying whether cell viability and the ability to survive desiccation and treatment
with disinfectants and lysozyme is affected if a larger region in the KL loci is knocked out.

38. Page | 27
Chapter 2: Materials and Methods
2.1 Bacterial strains used in this study
The details of bacterial strains used in this study can be found in Table 2.1.
2.2 Bacterial growth media, buffers and solutions
All stock solutions for media, solutions, buffers and gels were formulated following
manufacturers’ instructions and are described in Table 2.2. All media used for bacterial growth
were sterilised by autoclave. When antibiotics were added to growth medium for selection,
the growth medium was first autoclaved and then cooled to approximately 45 °C before
addition of the required filter sterilised stock solutions of antibiotic at the correct
concentration. This study used filter sterilised stock solutions of Ery 10 mg/mL, gentamicin
(Gent) 25 mg/mL, ampicillin (Amp) 100 mg/mL, tetracycline (Tet) 4 mg/mL, chloramphenicol
(Cml) 25 mg/mL, benzalkonium chloride (BAK) at 0.004% and chlorhexidine (CHG) at 0.008%.
Media that were used to culture A. baumannii and Escherichia coli were Luria–Bertani (LB)
broth, LB agar, Mueller–Hinton (MH) broth and MH agar.
For antibiotic selection, Ery 25 µg/mL, Gent 16 µg/mL, Amp 100 µg/mL, Tet 12 µg/mL and Cml
25 µg/mL were added as required, all of which were purchased from Sigma. For blue and white
colony selection, filter sterilised 1.25 µg/mL of 5-bromo-4-chloro-3-indoyl-β-D-galacto-
pyranoside (X-gal) and 1.25 µg/mL of isopropyl-β-D-galactopyranoside (IPTG) were added to
molten agar. Both X-gal and IPTG were purchased from Sigma. The LB agar was cooled to
approximately 45 °C before the IPTG and X-gal were added.

39. Page | 28
Table 2.1: Bacterial strains used in this study
Bacterial strains Genotype
Point of origin or
reference
Acinetobacter baumannii
ATCC 17978
Non-international clone;
meningitis isolate, capsular type
KL3
(Smith et al., 2007)
17978_∆cps A. baumannii ATCC 17978 from
gnaA to gtr9 removed
Jennifer Singh
17978_∆cps2 A. baumannii ATCC 17978 from
fkpA to lldP removed
This study
Escherichia coli DH5α Fᶲ80 lacZ ∆M15 ∆(lacZYA–argF)
U169 recA1 endA1 hsdR17(rK
-
,
mk
+
) phoA supE44 thi-1 gyrA96
relA1λ-
(Hanahan, 1983)

40. Page | 29
Table 2.2: Growth media, buffers and solutions
Media
Luria–Bertani broth
1 L
1% NaCl
1% tryptone
0.5% yeast extract
Adjusted to a pH of 7.5
10 g of agar was added to make LB agar in dH2O
Mueller–Hinton broth
OXOID
Milli-Q water was added to a final volume of 1 L
Mueller–Hinton agar
OXOID
Milli-Q water was added to a final volume of 1 L
Yeast extract peptone
dextrose broth
2% peptone
1% yeast extract
2% glucose in dH2O
Yeast extract peptone
dextrose agar
2% peptone
1% yeast extract
2% glucose
2% of volume is the amount add of agar in dH2O
Buffers and solutions

41. Page | 30
Tris-acetate
ethylenediaminetetra
acetic acid buffer (50×)
24.2% (w/v) Tris base
50 mM ethylenediaminetetra acetic acid (EDTA)
5.7% (v/v) glacial acetic acid
Adjusted to a pH of 8.0 in dH2O
GelRed nucleic acid
stain (Biotium)
0.03% GelRed
33 µM NaCl
in dH2O
Phosphate buffered
saline (PBS)
0.8% (w/v) NaCl
0.14% (w/v) Na2PO4
0.02% (w/v) KCl
0.024% (w/v) KH2PO4
Adjusted to a pH of 7.4 in dH2O
Sodium dodecyl sulfate
(SDS)-PAGE running
buffer
0.3% Tris base
1.44% glycine
0.1% SDS
dH2O was added to a final volume of 1 L
Fixative for SDS-PAGE
1 L
25% isopropanol
7% acetic acid
68% dH2O
Alcian blue stain 99.999% fixative solution for SDS-PAGE
0.001% Alcian blue

42. Page | 31
Transformation Factor
B 1 (TBF1)
30 mM potassium acetate
100 mM potassium chloride
10 mM calcium chloride
50 mM manganese chloride
15% glycerol
Dissolved in Milli-Q water, adjusted to pH of 5.8 with acetic
acid and filter sterilised with 0.2 µm filter
Transformation buffer
B 2 (TBF2)
10 mM 3-(N-morpholino) propanesulfonic acid (MOPS)
75 mM calcium chloride
10 mM potassium chloride
15% glycerol
Dissolved in dH2O, and filter sterilised with 0.2 µm filter
Gels
Agarose gel 0.8-1% (w/v) agarose
0.5× TAE buffer
in dH2O

43. Page | 32
20% SDS-PAGE gel 0.7 mL of in dH2O
6.6 mL 30% acrylamide
2.5 mL 1.5 M Tris (pH 8.8)
100 µL 10% SDS
100 µL 10% ammonium persulfate (APS)
4.0 µL tetramethylethylenediame (TEMED)
0.1% Stacking gel for
SDS-PAGE
3.4 mL of in dH2O
0.83 mL 30% acrylamide mix
0.63 mL 10% M Tris (pH 6.8)
30 µL 10% SDS
30 µL 10% APS
5 µL TEMED

44. Page | 33
2.3 Bacterial storage and growth conditions
Bacterial cultures were grown overnight in either LB or MH broth at 37 °C in a gyratory shaker
at 200 rpm. Bacterial cultures grown on solid LB agar media were incubated overnight in a
Laboro incubator (Townson and Mercer) at 37 °C, unless stated differently. Antibiotics were
added to broth and solid media before inoculation of bacterial cultures. For short-term
storage, culture plates were kept at 4 °C. For long-term storage of cultures, single colonies
were isolated and suspended in LB with 80% glycerol and kept at −80 °C.
2.4 Standard procedures
2.4.1 Purification of genomic DNA
Genomic DNA was purified and isolated using the Wizard® Genomic DNA Purification Kit
(Promega) following the manufacturer’s instructions. In brief, culture was streaked onto solid
media and incubated overnight at 37 °C. The culture was then removed and resuspended in
600 µL of nuclei lysis solution. This was incubated at 80 °C for 5 minutes before being cooled
to room temperature, after which 3 µL of RNase solution was added to the suspension,
inverted to mix and incubated for 15 minutes at 37 °C. The suspension was again cooled to
room temperature and 200 µL of protein precipitation solution was added, vortexed and the
suspension was then placed on ice for 15 minutes. The suspension was centrifuged at
13,500 rpm for 3 minutes using a Dynamica Velocity 13µ Minifuge. The supernatant was
transferred to a sterile 1.5 mL microfuge tube with 600 µL of room-temperature isopropanol,
which was inverted to mix. The DNA was removed with a glass rod and re-suspended in 600
µL of 70% ethanol. This was inverted to mix and then centrifuged for 13,500 rpm for 2 minutes.

45. Page | 34
Ethanol was aspirated and the pellet was left to dry for 15 minutes. Finally, the pellet was re-
hydrated in 100 µL of rehydration solution overnight at 4 °C.
2.4.2 Purification and isolation of plasmid DNA
The Bioline ISOLATE II Plasmid Mini Kit was used to isolate plasmid DNA following the
manufacturer’s instructions. In brief, 10 mL of overnight culture was pelleted by centrifugation
using a Hermle Z383K (4,500 rpm for 5 minutes at room temperature) where the supernatant
was discharged. The pellet was then re-suspended with Resuspension Buffer P1 by pipetting
up and down. Then, 500 µL of Lysis Buffer P2 was added and the 1.5 mL microfuge tube was
inverted 8 times and then incubated for 5 minutes, after which 600 µL of Neutralising Buffer
P3 was added, mixed by inversion 8 times and centrifuged at 11,000 rpm for 10 minutes at
room temperature using a Dynamica Velocity 13µ Minifuge.
The total of 750 µL of supernatant was pipetted into an ISOLATE II Plasmid Mini Spin Column,
which was already placed inside a 2 mL collection tube. This was centrifuged at 11,000 rpm
for 1 minute at room temperature using a Dynamica Velocity 13µ Minifuge. If there was
remaining supernatant from the sample, this process was repeated. Flow through was
discarded and 500 µL of preheated 50 °C Wash Buffer PW1 was added to the Mini Spin Column
and centrifuged (at 11,000 rpm for 1 minute at room temperature using a Dynamica Velocity
13µ Minifuge). Flow through was discarded and then a wash step with 600 µL of Wash Buffer
PW2 and centrifugation (at 11,000 rpm for 1 minute at room temperature using Dynamica
Velocity 13µ Minifuge). Flow through was discarded and then centrifuged for 2 minutes. The
ISOLATE II Plasmid Mini Spin Column was placed in a fresh 1.5 mL microcentrifuge tube with

46. Page | 35
addition of 50 µL of Ellusion Buffer P preheated to 70 °C and incubated for 2 mins. This was
centrifuged so that the purified plasmid would move into the fresh 1.5 mL microcentrifuge
tube. The DNA concentration was determined with a Thermo Scientific NanoDropTM
1000
Spectrophotometer (Biolab) and stored at 4 °C.
2.4.3 Polymerase chain reaction
Oligonucleotides used for polymerase chain reaction (PCR) in this study were synthesised by
SIGMA-ALDRICH (Table 2.3). All PCRs were performed using a 5
PRIME or GeneProTM
Thermal
cycler in 0.5 mL thin-walled tubes. Template DNA was either purified plasmid DNA, purified
chromosomal DNA, purified PCR products or whole cells (Table 2.4).
2.4.3.1 Standard polymerase chain reaction
The DNA polymerases Velocity, MangoTaqTM
and Econotaq were used in this study to amplify
different products. Velocity was used to amplify PCR products used for cloning into vectors as
well as for nested PCR for homologous recombination. Econotaq was used to amplify products
for cloning into pGEM®-T Easy vectors. Econotaq and MangoTaqTM
were used for all other PCR
amplifications. PCRs using Velocity DNA polymerase were made to a final volume of 50 µL,
which contained 200 ng of the forward and reverse primer in total, 200 ng of the template
DNA, 0.2 mM of dNTPs (Promega), 5 units of Velocity DNA polymerase (Bioline), 1 × Velocity
buffer and sufficient Milli-Q water to reach the final volume. Polymerase chain reactions using
Econotaq were made to a final volume of 20 µL, which contained 200 ng of the forward and
reverse primer in total, 200 ng of the template DNA, 0.2 mM of dNTPs (Promega), 1 × Econtaq

47. Page | 36
Table 2.3: Primers used in this study
Name Forward primer (5’–3’) Reverse primer (5’–3’)
Reference or
source
Primers used for creation and sequencing of ∆cps2
ERY_nol CTTAAAGAGTGTGTTGAT
AG
ATAGAATTATTTCCTCCCG This study
CPS2_ufr CAGTGTACTGTTTGCTGG CTATCAACACACTCTTAA
GATGAGTAAAGCCTTACC
C
This study
CPS2_dfr CGGGAAGGAAATAATTC
TATATGCTCAATATGTGG
C
GCTGCGGTAATGTCTGG This study
CPS2_ALL GGAATGACCTGGTTAAGC CCTGCGGTATTGGTCG This study
ERY_Read_Out CAGTTTCATCAACCAATG GGTTGAGTACTTTTTCACT
CG
Felise Adams
Primers used for the first step in the OAV system
pW_Ori_OAV GAAACTGGCAGCGAAGA
ATG
CACCCTTATCTATAAACAC
CCGAACAGGCTTATGTCA
ATTCG
This study

48. Page | 37
Name Forward primer (5’–3’) Reverse primer (5’–3’)
Reference or
source
Gent_OAV GTGTTTATAGATAAGGGT
GCGAATTGACATAAGCCT
GTTCGG
GCTTGAACGAATTGTTAG
GTGG
This study
All_OAV_pWgen
t
GAGA GCATGC
GCGAAGAATGAAGATTG
GAGA GCATGC
CGAATTGTTAGGTGGCGG
This study
pW-
Ori_pPR2274_V
2
CAGACGATGCAAAACGCA
AGATC
CCGAACAGGCTTATGTCA
ATTCGGCCGAAAAAAGAC
AATGACC
This study
M13 GTAAAACGACGGCCAG CAGGAAACAGCTATGAC Promega
All_OAV_pWgen
t_V2
GAGA GCATGC
GATCGGGGCTTACTTACT
G
GAGA GCATGC
CGAATTGTTAGGTGGCGG
This study
Gent_pPR2274_
V2_F
GGTCATTGTCTTTTTTCG
GCCGAATTGACATAAGCC
TGTTCGG
This study
AIS_2565 TGGCTCGATATTCAACGT
CA
TAACAGCAAACCACCACC
AA
Bart Eijkelkamp
Gent_Read_Out GCAGATTACGGTGACGAT
CC
CTGCTTGGATGCCCGAGG
CATAG
Felise Adams

49. Page | 38
Name Forward primer (5’–3’) Reverse primer (5’–3’)
Reference or
source
pW_Ori_sphI_R NA GAGAGCATGCGCCGAAA
AAAGACAATGACC
This study
Tet_xbaI GAGATCTAGAGGGGTTCC
GCGCACATTTCC
GAGATCTAGACAGTTCTC
CGCAAGAATTGATTGG
This study
pwh_tet_check GCGTTGATGCAATTTCTA
TG
GAAGCTGTCCCTGATGGT
C
Felise Adams
ppR2274_check CGAGAGCAAACTACCTCA
TAC
GCGAGTCAGTGAGCGAG
G
This study
PWH_ORI_NsiI GAGA ATGCAT
CGACCACGCTGATGAGCT
TTACCG
This study
PWH_ORI_XbaI GAGA TCTAGA
TTTTCACCGTCATCACCGA
AACGC
This study
pW_ori_check CCCCGATTTTATTGGGTA
CATTAGAG
This study
Mini-F_ori CCTGAAAAAACTTCCCTT
GGG
GGGATAACTTTGTGAAAA
AACAGCGGAAATGTGCG
CGGAACCCC
This study

50. Page | 39
Name Forward primer (5’–3’) Reverse primer (5’–3’)
Reference or
source
TET_v2_F GCTGTTTTTTCACAAAGTT
ATCCCGGGGTTCCGCGCA
CATTTCC
This study
ALL_mini-F_tet GAGAATGCATCCCTTGGG
GTTATCCACTTATCC
GAGAATGCATGCAAGAAT
TGATTGGCTCCAATTC
This study
Note: Bold primer sequence is assigned to overlapping sequence for nested PCR.

51. Page | 40
Table 2.4: Plasmids used in this study
Plasmid Characterisation
Point of origin or
reference
pATO4 pMMB67EH with RecAb system, TetR
(Tucker et al., 2014)
pPR2274 pCRG16; SmaI site in mini-F repE gene
removed (CCCGGG to CCCGAG), AmpR
, CmlR
(E.
coli), Cyhs
(yeast)
(Liu et al., 2017)
pWH1266 AmpR
, TetR
, fusion of pBR322 and pWH1277
using PvuII sites
(Hunger et al., 1990)
pWH1266 + 2006
+ Gent
AmpR
, TetR
, fusion of pBR322 and pWH1277
using PvuII site, with GentR
and ATCC 17978
A1S_2006
Felise Adams
pGEM-T easy
vector
AmpR
, ‘T’ base overhang cloning vector Promega
pGEM-T_ Gent GentR
cartridge inserted with TA cloning This study
pPR2274 _Tet TetR
cloned in the XbaI site in pPR2274 This study
pW_2274 pCRG16; SmaI site in mini-F repE gene
removed (CCCGGG to CCCGAG), AmpR
, CmlR
(E.
coli), Cyhs
(yeast), pW ori inserted from XbaI to
NsiI site
This study

52. Page | 41
buffer (Lucigen), 5 units of Econotaq polymerase (Lucigen) and sufficient Milli-Q water to
reach the final volume.PCRs using MangoTaqTM
were made to a final volume of 50 µL, which
contained 50 mM of MgCl2, 0.2 mM of dNTPs, 100 ng of the forward and reverse primer in
total, 200 ng of the template DNA, 5 × MangoTaqTM
coloured buffer and sufficient Milli-Q
water to reach the final volume.
The routine PCR cycling conditions for purified DNA samples comprised an initial f denaturing
step at 94 °C for 5 minutes, 30 cycles of a second denaturing step at 94 °C for 30 seconds, an
annealing step at 55 °C for 30 seconds and an elongation step at 72 °C for 1 minute per Kb.
This resulted in a final extension step at 72 °C for 10 minutes. Results of PCR reactions were
visualised by agarose gel electrophoresis.
2.4.3.2 Colony polymerase chain reaction
For colony PCR, single colonies were grown on LB plates with selection, picked with a pipette
and re-suspended in 40 µL of milli-Q water. For the PCR reaction, 8 µL of the colony sample
was used as template DNA. The PCR conditions followed those described in Section 2.4.3.1.
2.4.3.3 Nested polymerase chain reaction
For nested PCR, PCR products were used as template DNA and followed the standard PCR
conditions described in Section 2.3.3.1.

53. Page | 42
2.4.4 Agarose gel electrophoresis
Purified plasmid DNA and amplified PCR products were subjected to electrophoresis on
horizontal gels using routine molecular biological methods (Sambrook and Russell, 2001).
Agarose gels (0.8–1%) were used for visualising DNA products ranging from 0.4 to 4.5 Kb in
length (Table 2.2). Briefly, gels were cast for approximately 20 minutes before the DNA and
loading dye were loaded. To each sample, 0.4 µL per µL of sample DNA of either Gel Loading
Dye purple 6× (Biolabs) or Blue/Orange 6× Loading Dye (Promega) was added and 4 µL of
Hyperladder 1 (Bioline) was used as a molecular weight marker to identify DNA sizes ranging
from 0.2 to 10 Kb in length (Appendix 2). Electrophoresis was completed in 0.5× TAE buffer at
100 volts and stopped after approximately 30 minutes or when the loading buffer dye was
seen to have run approximately three quarters down the gel. The gel was removed from the
tank and stained in Biotum GelRed nucleic acid stain for 15 minutes (Table 2.2). The DNA
fragments were visualised using the Bio Rad Gel DocTM
EZ imager.
2.4.5 Purification of polymerase chain reaction products
DNA products amplified from PCR reactions (Section 2.3.3.1) were purified by using the
Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s
instructions. Briefly, equal parts of Membrane Binding Solution and PCR amplification
products were mixed by pipetting. This was added to the SV Minicolumn, which was inserted
into the collection tube. The sample was incubated for 1 minute at room temperature and
then centrifuged at 13,500 rpm for 1 minute. Flow through was discarded and 700 µL of
Membrane Wash Solution added and centrifuged for at 13,500 rpm for 1 minute. Flow
through discarded and 500 µL of Membrane Wash Solution was added and then centrifuged

54. Page | 43
at 13,500 rpm for 2 minutes. Flow through was discarded and the sample was then centrifuged
13,500 rpm for 2 minutes. The SV Minicolumn was then transferred to a sterile 1.5 mL
microcentrifuge tube where 20 µL pre-heated 60 °C Nuclease-Free water was added and
incubated for 1 minute. This was then centrifuged at 13,500 rpm for 1 minute and the DNA
concentration was determined by a Thermo Scientific NanoDropTM
1000 spectrophotometer
(Biolab). The final sample was stored at 4 °C.
2.4.6 Digestion of DNA with restriction endonucleases
All DNA restriction enzymes were used with the corresponding buffer solutions purchased
from New England Biolabs (Genesearch, Australia), following the manufacturer’s instructions.
PCR and plasmid DNA were digested in 30 µL volumes for 2–4 hours at 37 °C (Table 2.4). When
double digestion was required, compatible buffers were used to achieve the optimal cutting
activity between the two enzymes.
2.4.7 Adenosine treatment of polymerase chain reaction products
For TA cloning using the pGEM-T easy vector, purified PCR products were treated with
adenosine to allow ligation with the thymine overhangs of the linear form of the pGEM-T easy
vector (Section 2.3.5). Purified PCR products were treated with 5 units of Econotaq (Lucigen)
DNA polymerase, 1× Econotaq (Lucigen) buffer, 0.2 mM of dNTPs (Promega) and sufficient
Milli-Q water to reach a final volume of 50 µL. Reactions were incubated for 15 minutes at
37 °C.

55. Page | 44
2.4.8 Ligation of DNA
Ligation reactions were performed with restriction endonucleases digested products in a 3:1
insert to vector ratio in a final volume of 20–30 µL, unless otherwise stated. The ligations
comprised 2 Weiss units of T4 DNA ligase and 1× T4 DNA ligase reaction buffer (New England
Biolabs). Ligation reactions were incubated overnight at 4 °C.
2.4.9 DNA sequencing
All sequencing undertaken in this thesis was performed by the Australian Genome Research
Facility (AGRF) in a final volume of 12 µL. Each sequencing reaction contained 250 ng of
template DNA, 100 ng of the selected primer and sufficient Milli-Q water to reach a final
volume of 12 µL in a 1.5 mL microfuge tube. The DNA sequence was analysed using the
program SequencherTM
4.1.4 (Gene Codes Corp). To find any mutations in the sequenced DNA,
the DNA was aligned against consensus sequence CP012004.1 acquired from the National
Center for Biotechnology Information (NCBI) database.
2.4.10 Transformation of Escherichia coli
2.4.10.1 Preparation of chemically competent cells
Overnight cultures of E. coli DH5α were diluted into 1 into 20 freshly warmed LB media and
incubated for one hour at 37 °C in constant agitation at 200 rpm. Cells were then further
diluted by transferring 200 µL of culture into 200 mL of freshly warmed LB media. The culture
was then incubated at 37 °C with continuous agitation at 200 rpm until the cellular density
reached an OD600 of 0.6. The culture was then divided between sterile chilled centrifuge tubes
and incubated for 5 minutes on ice. The culture was then centrifuged using a Hermle Z383K

56. Page | 45
centrifuge at 15,000 rpm for 10 minutes at 4 °C to pellet the cells. The supernatant was
discarded, and the cells were resuspended in 40 mL of TFB1 (Table 2.3) and incubated for 10
minutes on ice. Then the culture was centrifuged using a Hermle Z383K centrifuge at 15,000
rpm, for 10 minutes at 4 °C and the supernatant was discarded. The pelleted cells were
resuspended in 3.2 mL of TFB2 (Table 2.3) and incubated on ice for 15 minutes. The
resuspended cells were aliquoted into 100 µL sterile chilled microfuge tubes before being
stored at −80 °C for future use.
2.4.10.2 Transformation of competent Escherichia coli cells
Chemically competent E. coli DH5α cells were used for transformation with plasmid DNA
(Section 2.3.10.1). After the cells were removed from the freezer and thawed on ice for
15 minutes, 150 ng of plasmid DNA or ligated DNA products of interest (Section 2.3.8) was
added to the thawed cells, which were then tapped to mix and incubated on ice. Cells were
then heat shocked for 90 seconds at 42 °C and then immediately placed on ice for 15 minutes
for recovery. Next, 500 mL of sterilised LB broth was added to the cells and they were
incubated for one hour at 37 °C under constant agitation at 200 rpm. The cells were
centrifuged at 13,500 rpm for 1 minute using a Dynamica Velocity 13µ Minifuge. Of the
resulting supernatant, 100 µL was plated onto selective media for isolation of ligated plasmids.
The remaining supernatant was partially removed and the pellet was resuspended in 30 µL of
the supernatant and then spread onto plates containing selective media. The resulting plates
were incubated overnight at 37 °C.

57. Page | 46
2.4.11 Electroporation of Acinetobacter baumannii
2.4.10.1 Preparation of electrocompetent Acinetobacter baumannii cells – A
A. baumannii cells were isolated from single colonies and grown in LB broth overnight. The
overnight culture was then diluted to 1 into 4 with LB broth that included selection and IPTC
at 2 mM. The cells were incubated at 37 °C in a gyratory shaker to an OD600 of 0.04 (early log
phase). The cells were then centrifuged at 4,500 rpm for 10 minutes at −2°C using a Hermle
Z383K centrifuge and the supernatant was discarded. The pelleted cells were then
resuspended in 20 mL of 10% glycerol to wash them and were centrifuged again at 4,500 rpm
for 10 minutes at −2°C using a Hermle Z383K centrifuge. The supernatant was discarded and
the cells were washed again by being resuspended in 10 mL of 10% of glycerol before another
centrifugation at 4,500 rpm for 10 minutes at −2°C using a Hermle Z383K centrifuge. The
supernatant was discarded and the cells were resuspended in 250 µL of 10% glycerol. The cells
were used immediately.
2.4.11.2 Preparation of electrocompetent Acinetobacter baumannii cells – B
A. baumannii wild type (WT) cells were isolated from single colonies and grown in 10 mL of LB
broth overnight at 37 °C. The overnight culture was then diluted to 1 into 100 in 60 mL of LB
broth, which was incubated at 37 °C in a gyratory shaker until an OD600 of 1 was reached. Cells
were then centrifuged at 4,500 rpm for 10 minutes at −2°C using a Hermle Z383K centrifuge
and the supernatant was discarded. Pelleted cells were then resuspended in 10 mL of chilled
10% glycerol to wash and re-centrifuged at 4,500 rpm for 10 minutes at −2°C using a Hermle
Z383K centrifuge, after which the supernatant was discarded. This was repeated three times.
Cells were resuspended in 600 µL of 10 % glycerol and then aliquoted into 150 µL for
electroporation. The cells were used immediately.

58. Page | 47
2.4.11.3 Electroporation of electrocompetent A. baumannii cells – A
For electroporation, purified PCR amplified DNA product (approximately 250 ng) was added
to electro-competent A. baumannii cells (Section 2.4.11.1) and incubated for 5 minutes on ice.
Cells were electroporated at 1.8 kV, 200 Ω at 25 µF in a Gene Pulser ® II (Bio-Rad). Cells were
placed on ice for 5 minutes and then 4 mL of LB broth was added with 2 mM of IPTG and the
cells transferred to a gyratory shaker for 1 hour at 37 °C. The cells were then centrifuged at
4,500 rpm for 2 minutes at −2 °C. Pelleted cells were resuspended in 500 µL of LB broth and
plated onto LB agar with appropriate antibiotic selection before being incubated overnight at
37 °C. Single colonies were then selected and streaked onto fresh Ery 10 LB plates, which were
then screened further.
2.4.11.4 Electroporation of electrocompetent A. baumannii cells – B
Purified plasmid product was added at 100 ng to electro-competent A. baumannii cells for
electroporation (Section 2.4.11.3). The cells were incubated for 10 minutes at room
temperature before they were electroporated at 1.8 kV, 200 Ω at 25 µF in a Gene Pulser® II
(Bio-Rad). Then, 600 µL of LB broth was added to the cells for recovery in a gyratory shaker
for 1 hour at 37 °C. Next, the culture was centrifuged at 13,500 rpm for 1 minute at room
temperature using a Dynamica Velocity 13µ Minifuge. Cells were resuspended in 100 µL of LB
broth, plated onto LB agar with Amp 200 and Amp 150, and incubated overnight at 37 °C.
Single colonies were then selected and streaked onto fresh Amp 200 LB plates, which were
then screened further.

59. Page | 48
2.5. Characterisation of Acinetobacter baumannii strains
2.5.1 Analysis of growth
2.5.1.1 Growth curve
From a single colony, A. baumannii strains were incubated overnight in 10 mL of MH broth in
a gyratory shaker at 200 rpm. The overnight cells were diluted 1 into 100 in 35 mL of fresh MH
broth and incubated at 37 °C in a gyratory shaker at 200 rpm. The initial OD600 reading was
recorded after dilution and an OD600 reading was then recorded every hour after for a total of
8 hours until the OD600 reading reached 1.
2.5.1.2 Colony morphology
To identify if colony morphology had changed due to capsule knockouts in the strain ATCC
17978, single colonies of A. baumannii strains were streaked across LB agar plates and
incubated overnight at 37 °C. Colonies were then observed under a motic BA200 educational
routine laboratory microscope (compound).
2.5.2 Lysozyme assay
From a single colony, A. baumannii strains were incubated overnight in 10 mL of LB broth at
200 rpm. Overnight cultures were then diluted to 1 into 40 in 10 mL of fresh LB broth and
incubated at 37 °C in a gyratory shaker at 200 rpm until an OD600 of 0.6 was reached. The
bacterial culture was further diluted to an OD600 of 0.1 and then further diluted to an OD600 of
0.05; cells were kept on ice throughout this dilution process. To count initial concentrations
of the A. baumannii strains, −2, −3 and −4, dilutions were plated. For the lysozyme treatment,
50 µL of lysozyme (10 mg/mL) was added to 450 µL of bacterial culture and incubated for

60. Page | 49
1 hour at 37 °C in a gyratory shaker at 200 rpm. A negative control was prepared by adding
50 µL of Milli-Q water to 450 µL of bacterial culture and incubating this for 1 hour at 37 °C in
a gyratory shaker at 200 rpm. Aliquots of 100 µL were then plated on LB agar plates and
incubated overnight at 37 °C.
2.5.3 Desiccation assay
From a single colony, A. baumannii strains were incubated overnight at 200 rpm in 10 mL of
LB broth. The overnight culture was then diluted to 1 into 100 in 10 mL of LB broth and
incubated at 37 °C in a gyratory shaker at 200 rpm until an OD600 of 0.15 was reached. The
cells were centrifuged at room temperature for 5 minutes at 4,500 rpm using a Hermle Z383K
centrifuge. The supernatant was discarded and the pellet was resuspended in 5 mL of sterile.
Next, 25 µL of resuspended bacterial inoculum was pipetted into a 96-well flat bottom
polystyrene plate at room temperature. At each time point, cells were re-hydrated by
incubating in 100 µL of sterile for 30 minutes. Cells were serially diluted and plated onto LB
agar plates, which were then incubated overnight at 37 °C and bacterial colonies counted
(Chin et al., 2018).
2.5.4 Sensitivity to disinfectants
From a single colony, A. baumannii strains were incubated overnight at 200 rpm in 10 mL of
LB broth. The overnight culture was then diluted to 1 into 100 in 10 mL of LB broth. Cells were
incubated until an OD600 of 0.15 was reached. The 990 µL of cell culture was then aliquoted
into microfuges tubes with the addition of 10 µL of CHG 0.008%, or 10 µL of BAK 0.004% or 10
µL of sterilised water. The cells were then incubated for 30 minutes at 37 °C in a gyratory

61. Page | 50
shaker at 200 rpm. The treated cells were then serially diluted with a microtiter plate and
plated on LB agar plates. These were incubated overnight at 37 °C and enumerated the next
day.
2.5.5 Characterisation of capsule material
2.5.5.1 Cell preparation for capsule isolation
From a single colony, A. baumannii strains were incubated overnight at 200 rpm in 10 mL of
LB broth. Overnight cultures were further diluted to 1 into 40 in 20 mL of LB broth and then
incubated at 37 °C in a gyrating shaker at 2000 rpm until an OD600 of 1 was reached. Cells were
pelleted by centrifugation at 4,500 rpm for 5 minutes at 5 °C using a Hermle Z383K centrifuge
and the supernatant was discarded. The pellet was resuspended in 1 mL of lysis buffer (Table
2.3) with 25 µL of 10 mg/mL hen’s egg derived lysozyme (Sigma), 25 µL of RNAse (25mg/mL)
and 20 µL (25mg/mL) of DNAse. Cells were vortexed and incubated for one hour at 37 °C. Cells
were then crushed using a Cell Disrupter (Constant Systems LTD) and approximately 500 µL of
sample was recovered. To the recovered sample, 10 µL of DNAse and 10 µL of RNAse was
added and the samples incubated for 30 minutes at 37 °C. Next, 20 µL of 10% sodium dodecyl
sulfate (SDS) was added to each sample and incubated at 37 °C for 30 minutes. Samples were
processed by boiling for 5 minutes at 100 °C, after which 1 µL of proteinase K (25 mg/mL) was
added and incubated for 1 hour at 60 °C. Samples were then frozen at −20°C for later use.
2.5.5.2 SDS-PAGE and staining
Frozen samples were thawed and suspended in 1× SDS-PAGE buffer (Table 2.2) in a 1 to 1
ratio. The samples were then heated to 37 °C for 20 minutes. Next, 20 µL of the sample was

62. Page | 51
added to the SDS-PAGE well with 10 µL of Bio-Rad Precision Plus ProteinTM
Dual Colour
Standard to visual size. The gel was run at 90 volts for approximately 12 hours. The gel was
then submerged in fixative solution (Table 2.2) for 1 hour, removed and then submerged in
Alcian blue stain with shaking overnight. The gel was de-stained for 6 hours in fixative solution
(Table 2.2) and imaged using the Bio Rad Gel DocTM
EZ imager.

63. Page | 52
Chapter 3: Results
3.1 Introduction
A. baumannii is an important nosocomial pathogen due to the difficulty of treating infections
and decontaminating hospitals (Harding et al., 2017). These factors have resulted in
A. baumannii being categorised by the World Health Organization as priority 1 for research
into new antibiotic design (WHO, 2017). One of the factors contributing to the success of
A. baumannii in the nosocomial setting is its ability to express a capsule (Harding et al., 2017);
see Section 1.3. A. baumannii’s capsule is known to aid in colonisation, desiccation resistance,
biofilm formation and resistance to the host’s immune system (Powers and Trent, 2018,
Harding et al., 2017, Wang-Lin et al., 2017). To date, there has been no direct analysis of
different capsule types on these virulence traits without background strain genetics
influencing the results. Therefore, this study aims to create an OAV that can incorporate
capsule regions from different serotypes for direct comparison into the same background. This
was chosen as the method of cloning because the cps region in A. baumannii is very large,
which would have been difficult to clone with conventional methods. The OAV also allows for
genetic manipulation once created.
As a first step, a ∆cps derivative, ∆cps2, was be constructed and phenotypically analysed.
∆cps2 was designed as the isogenic host for direct comparison of the OAV system because the
entire KL region has been deleted. ∆cps2 also has the capsular polysaccharide export genes
(wzc, wza and wzb) and initiating transferases removed. These are known to vary between KL
regions in different strains of A. baumannii (Figure 1.1). The smaller deletion mutant, ∆cps,
retains the CPS export system (wzc, wzb and wza), initiating transferase (itrA2), and some

64. Page | 53
intermate and biosynthesis sugars (galU, Ugd, gpi, gnr1 and pgm) (Figure 3.1). Because the
export system, initiating transferases, intermate and biosynthesis sugars vary between A.
baumannii strains, this affects the direct comparison of the K structures produced in this
model (Figure 1.1 (A)). It was decided to construct a larger deletion in the cps region for the
isogenic model. Phenotypical analysis comparing ∆cps to ∆cps2 determined whether deleting
a larger region in the KL locus in ATCC 17978 affects cell viability (see Section 3.3).
The strain A. baumannii ATCC 17978 was chosen to the isogenic model for the OAV system
because it contains few resistance genes. This enables more diversity in the choice of selective
markers that can be used in the OAV design. It also enables existing selective genes in the
vector pPR2274 to be used (Appendix 6). This means that the isogenic model can be selected
for when it has accepted the OAV, which aids in the initial stages of screening.
The OAV protocol was adapted from an engineered yeast–E. coli shuttle vector designed by
Raymond et al. (2002). The vector was used to clone whole Pseudomonas aeruginosa O-
antigen gene clusters from sheared DNA using the double-strand repair pathway in
Saccharomyces cerevisiae (Raymond et al., 2002). This was then expanded by Liu et al. (2017)
who developed an OAP to simultaneously create several operon clones through the use of
multiple overlapping PCR products that underwent homologous recombination by the DNA
repair pathway in yeast (Liu et al., 2017). Liu et al.’s (2018) OAP system allows rapid
simultaneous cloning of large regions of genetic material in the same isogenic background for
direct functional analysis in operon gene clusters (e.g. the KL loci in A. baumannii). This allows
the direct analysis of different capsule types (K structures) on desiccation, antimicrobial
resistance and immune evasion without having additional genes affecting the results.

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Figure 3.1: Cps gene region in ATCC 17978, ∆cps and ∆cps2 strains.
The strain names are indicated on the left as well as the capsule loci (KL) in Acinetobacter
baumannii. Horizonal arrows indicate genes and their direction of transcription.
Corresponding gene names are shown above the gene. Gene colour indicates homology as
both ∆cps and ∆cps2 are mutants of the parental strain ATCC 17978, the KL is located between
fkpA to lldP, shown in pink and purple respectively. The following genes are involved in CPS
export; wzc, wzb and wza. Nucleotide-sugar biosynthesis are encoded by; gna, dgaA and dcaC.
Genes involved in K unit processing are wzx and wzy, in yellow. Genes involved in glycan
modification through acetyl or acyl transferases are dcaB and atr2. Glycosyltransferase are
encoded by; gtr6, gtr7, gtr8 and gtr9. Initiating transferase is encoded by itrA2. Pathway
intermediates and biosynthesis of glucose derivates are encoded by; galU, Ugd, gpi, gne1 and
pgm (Kenyon and Hall, 2013). The gene deletions in acapsular strains ∆cps and ∆cps2 are
indicated by a grey dashed line. KL3 indicates the capsule structure produce by ATCC17978.
Gene size is not to scale.

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To use the OAP system in A. baumannii, the plasmid had to be adapted to contain an origin of
replication specific to Acinetobacter (Liu et al., 2017); see Section 3.4.1. The first step was to
construct the acapsular mutant ∆cps2, which was used as the isogenic model for the OAV
system. Phenotypic analysis of ∆cps2 first involved comparing ∆cps and ∆cps2 to determine
whether a larger knockout in the KL locus would affect cell viability (Section 3.3; Figure 3.1).
The second step of phenotypic analysis was to determine whether survival during desiccation,
resistance to disinfectants (BAK and CHG) and resistance to lysozyme were decreased when
the capsule was absent. The following chapter describes these results.
3.2 Construction of a cps deletion in Acinetobacter baumannii ATCC 17978
Homologous recombination was used to create the ∆cps2 ATCC 17978 derivative (see Sections
2.4.3.2, 2.4.5, 2.4.11.1 and 2.4.11.2). The two sites of homology were fkpa and lldP, which are
located on either side of the cps region (Figure 3.1). This method involved first amplifying the
fkpA and lldP genes and the antibiotic selection marker Ery. Amplification of the fkpA gene
involved using CPS2_ufr_F/R primers to amplify a region of 690 bp that was visualised using
electrophoresis on an agarose gel for a band of 700 bp, which was identified on the gel (Table
2.3; data not shown). Amplification of the lldP gene involved using CPS2_dfr_F/R primers to
amplify a region of 767 bp (Table 2.3; data not shown). This was visualised using
electrophoresis on an agarose gel for a band of 750 bp, which was identified on the (data not
shown). Amplification of the Ery cartidge used ERY_nol_F/R primers to amplify a region of 844
bp that was visualised using electrophoresis on an agarose gel for a band of 850 bp, which was
identified on the gel (Table 2.3; data not shown).

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Once the band sizes were identified at 700 bp for fkpA, 750 bp for lldP and 850 bp for Ery, they
were added as template DNA for the nested PCR in a ratio of 1:1:1 with the CPS2_ALL_F/R
primer to connect the three PCR products (Table 2.3). This was visualised using electrophoresis
on agarose gel to determine the band size of 2.3 Kb (data not shown). Once the band size was
determined at 2.3 Kb, the nested PCR product was purified and the amount of DNA was
measured. Next, 300 ng of the nested PCR product was electroporated at 1.8 volts into ATCC
17978. The electroporated cells were then plated on LB agar that contained Ery for selection
of resulting mutant colonies. Resulting colonies were then screened with colony PCR for the
nested product using CPS2_ALL_F/R with a corresponding band size of 2.3 Kb (data not
shown). After the initial identification of the 2.3 Kb region, the colony DNA was then sent to
AGRF for sequence validation using Ery_Read_Out_F/R, primers (Table 2.3; data not shown).
The resulting cps knockout strain was called ∆cps2 and contains the fkpA, Ery and lldP genes
in the cps region (Figure 3.1). ∆cps2 has 22 genes or 23 Kb in ATCC 17978 replaced with an Ery
cartridge of 844 bp (Figures 3.1 and 3.2). Phenotypic analysis of ∆cps2 involved comparing
∆cps2 with the WT strain ATCC 17978 WT, and the smaller capsule knockout mutant ∆cps
(Figure 3.1). Further phenotypic analysis involved comparison of ∆cps and ∆cps2 to determine
whether a bigger deletion in the cps region affects cell viability (Figure 3.1).
3.3 Characterisation of ∆cps2
A number of assays were used to analyse the impact of removal of the capsule in ATCC 17978.
Functional characterisation involved comparing ∆cps2 with both the WT ATCC 17978 and the
smaller knockout mutant ∆cps on growth rates, colony morphology, desiccation survival,
resistance to disinfectants and resistance to lysozyme.

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Figure 3.2: Homologous event in ATCC 17978 to create ∆cps2.
The genes fkpA and lldP are shown in pink and purple, respectively. The cps region is shown
in one block for simplicity and contains 22 genes (Figure 3.1). The thin blue arrows show the
replacement of the deleted cps region (23 Kb) in ∆cps2 with an Ery cartridge (8444 bp). The
∆cps2 mutant was created from ATCC 17978 (WT). The primers used for sequencing can be
seen in purple and are labelled CPS2_ALL_F/R (Table 2.3).