Download Bacterial viruses targeting multi- resistant Klebsiella

Bacterial viruses targeting multiresistant Klebsiella pneumoniae
and Escherichia coli
Harald Eriksson
©Harald Eriksson, Stockholm University 2015
ISBN 978-91-7649-123-2
Published papers are reproduced with the permission of the publisher.
Printed in Sweden by Holmbergs, Malmö 2015
Distributor: Department of Molecular Biosciences, the Wenner-Gren Institute
Populärvetenskaplig sammanfattning
Ett växande problem i dagens
samhälle är den ökade resistensen
hos bakterier mot antibiotika,
vilket i det långa loppet kan leda
till att tidigare milda infektioner
blir svårbehandlade och i vissa fall
dödliga. Forskning pågår på flera
fronter för att lösa detta problem;
från att minska användningen av
antibiotika och endast behandla
svåra infektioner, till att ny- eller
vidareutveckla ny antibiotika.
Dessutom sker det forskning på att
utveckla alternativa behandlingsmetoder
mot
bakteriella
infektioner, som inte är lika tidsoch
kostnadsintensiva
som
utvecklingen av ny traditionell
antibiotika.
Fokus i denna avhandling har varit
bakteriens
naturliga
fiende,
bakterieviruset, som i vissa fall
kan nyttjas i terapeutiskt syfte för
avdödandet av bakterier i
infektioner
hos
människan.
Genom att isolera bakterievirus
från miljöer där det sker en
kontinuerlig kamp om överlevnad
mellan bakterierna och dess virus,
kan vi ta del av den naturliga
genetiska variation som det finns
mellan dessa. Studierna i denna
avhandling har lett till att vi
isolerat ett flertal nya bakterievirus, primärt mot multiresistenta
Klebsiella pneumoniae men även
mot Escherichia coli. De isolerade
bakterievirusen
har
sedan
karaktäriserats, från evolutionära
och taxonomiska aspekter (artikel
I, II), till ingående analys av
arvsmassa, strukturella proteiner
och infektionsförlopp (artikel II).
Vidare har vi utvärderat hur en
sammansatt blandning av bakterievirus lyckas avdöda en större
mängd kliniska isolat av multiresistenta K. pneumoniae (artikel
III). Slutligen har vi studerat den
biologiska effekten av predation
på den bakteriella värden, genom
att studera ett kliniskt isolat av
multiresistent K. pneumoniae och
deras förmåga att producera
biofilm, deras tillväxthastighet och
virulens (artikel IV) efter att ha
utvecklat
resistens
mot
antagonistiska bakterievirus.
Abstract
The global increase in antibiotic
resistance levels in bacteria is a
growing concern to our society and
highlights the need for alternative
strategies to combat bacterial
infections.
Bacterial
viruses
(phages) are the natural predators
of bacteria and are as diverse as
their hosts, but our understanding
of them is limited. The current
levels of knowledge regarding the
role that phage play in the control
of bacterial populations are poor,
despite the use of phage therapy as
a clinical therapy in Eastern
Europe.
The aim of this doctoral thesis
is to increase knowledge of the
diversity and characteristics of
bacterial viruses and to assess their
potential as therapeutic agents
towards multi-resistant bacteria.
Paper I is the product of de novo
sequencing of newly isolated
phages that infect and kill multiresistant Klebsiella pneumoniae.
Based on similarities in gene
arrangement, lysis cassette type
and conserved RNA polymerase,
the creation of a new phage genus
within
Autographivirinae
is
proposed.
Paper II describes the genomic
and proteomic analysis of a phage
of the rare C3 morphotype, a
Podoviridae phage with an
elongated head that uses multi-
resistant Escherichia coli as its
host.
Paper III describes the study of
a pre-made phage cocktail against
125 clinical K. pneumoniae
isolates. The phage cocktail
inhibited the growth of 99 (79 %)
of the bacterial isolates tested. This
study also demonstrates the need
for common methodologies in the
scientific community to determine
how to assess phages that infect
multiple serotypes to avoid false
positive results.
Paper IV studies the effects of
phage predation on bacterial
virulence: phages were first
allowed to prey on a clinical K.
pneumoniae isolate, followed by
the isolation of phage-resistant
bacteria. The phage resistant
bacteria were then assessed for
their growth rate, biofilm
production in vitro. The virulence
of the phage resistant bacteria was
then
assessed
in
Galleria
mellonella. In the single phage
treatments, two out of four phages
showed an increased virulence in
the in G. mellonella, which was
also linked to an increased growth
rate of the phage resistant bacteria.
In
multi-phage
treatments
however, three out of five phage
cocktails decreased the bacterial
virulence in G. mellonella
compared to an untreated control.
List of papers
This doctoral thesis is based on the papers listed below, referred to in the text
by their associated Roman numerals.
I.
Eriksson, H., Maciejewska, B., Latka, A., MajkowskaSkrobek, G., Hellstrand, M., Melefors, Ö., Wang, JT.,
Kropinski, A.M., Drulis-Kawa, Z. and Nilsson, A.S.
A suggested new bacteriophage genus, “Kp34likevirus”,
within the Autographivirinae subfamily of Podoviridae.
Viruses 2015, 7, 1804-1822; doi:10.3390/v7041804
II.
Khan Mirzaei, M., Eriksson, H., Kasuga, K., HaggårdLjungqvist, E., Nilsson, A.S.
Genomic and proteomic analysis of ECORp10, a newly
characterized Podoviridae phage with C3 morphology.
PLoS ONE 2014, 9(12): e116294;
doi:10.1371/journal.pone.0116294
III.
Eriksson, H., Berta, D., Örmälä-Odegrip, A., Giske, G. C.,
Nilsson, A.S.
A novel phage cocktail inhibiting the growth of 99 βlactamase carrying Klebsiella pneumoniae clinical isolates in vitro
Manuscript.
IV.
Örmälä-Odegrip, A., Eriksson, H., Mikonranta, L.,
Ruotsalainen, P., Mattila, S., Hoikkala, V., Nilsson, A.S.,
Bamford, J.K.H. and Laakso, J.
Evolution of virulence in Klebsiella pneumoniae treated with
phage cocktails
Manuscript.
Table of contents
Introduction .................................................................................................. 9
Phages ......................................................................................................................... 9
Phage classification .............................................................................................. 9
Caudovirales taxonomy .................................................................................... 10
Phage infection ................................................................................................... 12
Phage evolution .................................................................................................. 13
Phage impact on bacterial hosts ........................................................................... 15
Phage–bacteria coevolution ............................................................................. 15
Bacterial defense systems ................................................................................ 16
Phage therapy .......................................................................................................... 22
The rise of antibiotic resistance....................................................................... 22
The history of phage therapy........................................................................... 23
Phage therapy today ......................................................................................... 24
Proteins as antimicrobial compounds ............................................................. 26
Enterobacteriaceae ................................................................................................. 27
Klebsiella spp. ..................................................................................................... 27
Escherichia coli ................................................................................................... 28
Aims.............................................................................................................. 29
Results and discussion ............................................................................. 30
Paper I ....................................................................................................................... 30
Paper II ..................................................................................................................... 32
Paper III .................................................................................................................... 33
Paper IV .................................................................................................................... 34
Concluding remarks and future perspectives ..................................................... 35
Acknowledgments ..................................................................................... 38
References .................................................................................................. 40
Abbreviations
Abi
carba
Cas
CRISPR
DNA
ECOR
ESBL
HGT
Kpn
LO
LPS
LIN
MS
MOI
PEG
RNA
RM
SEM
Sie
TA
TEM
abortive infection
carbapenem
CRISPR associated genes
Clustered Regularly Interspaced Short Palindromic
Repeats
deoxyribonucleic acid
Escherichia coli reference collection
extended spectrum β-lactamase
horizontal gene transfer
Klebsiella pneumoniae
lysis from without
lipopolysaccharide
lysis inhibition
mass spectrometry
multiplicity of infection
polyethylene glycol
ribonucleic acid
restriction modification
scanning electron microscopy
superinfection exclusion
toxin-antitoxin
transmission electron microscopy
Introduction
Phages
Bacteriophages, or phages for short, are viruses that infect and propagate
in bacteria. Phages are the most abundant biological entity on the planet, with
estimated amounts of up to 1031 in the biosphere (Hendrix et al., 1999). Out
of all isolated phages, 96 % are tailed phages that belong to the order
Caudovirales (Ackermann, 2001). Phages are ancient molecular machines
that are believed to have existed by the time bacteria, archaea and eukarya
diverged in the tree of life, based on the folding patterns of their proteins
(Ackermann, 1998). They are highly specific for their target bacterial strain;
this specificity is often as narrow as individual serotypes of a bacterial species.
The large biodiversity of viruses is reflected in but also driven by their hosts,
the prokaryotes and archaea. Phages can carry either RNA or DNA genomes
that are either single or double stranded, and they can be enveloped or nonenveloped and have varied morphologies.
Phage classification
The relatively simple and straight forward method of classifying
prokaryotes by their 16S rRNA (Woese and Fox, 1977) has no analogue in the
phage world (Hendrix et al., 1999). Phages traditionally have been
taxonomically classified based on the visualization of the free virion structure.
With advances in whole genome sequencing, attempts have been made to
classify phages in more detail. The mosaic-like structure of phage genomes
requires the use of more intricate methods to classify them, not only by
comparing the identities of individual genes but also the spatial placement of
the genes in the genome and the gene order (Hendrix et al., 1999).
The crystal structures of phage capsid proteins have revealed a conserved
three dimensional (3-D) structure, even though there is no or very low amino
acid sequence similarity between phage families. These tertiary structures
recur throughout the viral kingdom, e.g., between the capsid folds of
Podoviridae HK97, Myoviridae T4 and even in the eukaryote Herpes simplex
virus (Cardone et al., 2013). This structural conservation of phage proteins is
evident in several parts of the phage virion, from the assembly of the phage
9
head by the capsid proteins to the joining of the head and tail assembly by the
head to tail connector proteins and also in the setup and assembly of phage
tails. The phage tails consist of tape measure proteins, tail tube proteins, tail
terminator proteins and, in the case of Myoviridae, the tail sheath protein. At
the distal end, the host adsorption apparatus can be either a relatively simple
tail spike or a more complex baseplate with tail fibers, or both components
(Veesler and Cambillau, 2011).
The head to tail connector proteins exhibit large size and sequence
variations between phages, as with the 37-kDa Φ29 portal protein compared
to its 83-kDa P22 counterpart. Regardless of size variation, these compared
portal proteins assemble into an overall similar structure (Veesler and
Cambillau, 2011). The similarity of the tertiary structures of these viral
proteins might either be an indication of a common archaea/eukarya ancestry
for all viruses or evolutionary convergence (Veesler and Cambillau, 2011).
Caudovirales taxonomy
The phages of Caudovirales are classified into three distinct families (fig. 1)
based on their tail morphology. While the archetypical phage has an
icosahedral and symmetrical head, there is a variation with an elongated
morphology for the head structure, often referred to by a letter followed by a
numeral; as shown, A represents Myoviridae phages (fig. 1A), B represents
Siphoviridae (fig. 1B) and C is Podoviridae (fig. 1C). The numeral can range
from 1 to 3: 1 is a symmetrical head, 2 an elongated head with a length-towidth ratio ranging between 1 and 2, and 3 has a length-to-width ratio greater
than 2 (Bradley, 1967).
Podoviridae have a short and non-flexible receptor binding complex for
cell adhesion and DNA injection into the host cell, and their genome is
encoded by double-stranded DNA. Further subdivision of the Podoviridae
family comprises the Autographivirinae and the Picovirinae subfamilies; the
Autographivirinae-type phages carry a gene for a single-subunit RNA
polymerase and thus have the ability to transcribe their own genes, while the
Picovirinae are classified on the basis of their small genome size, special tail
vertex structure and DNA polymerase (Adriaenssens et al., 2015, Lavigne et
al., 2008). From recent classification studies, it has been shown that while
horizontal gene transfer (HGT) mechanisms are a contributing factor to the
genetic diversity and exchange of genetic material between phages,
evolutionary relationships can be readily observed between different phage
subfamilies and genera (Adriaenssens et al., 2015, Lavigne et al., 2009,
Lavigne et al., 2008).
10
The second Caudovirales family are the Siphoviridae, which have a
double-stranded DNA genome, and a long, flexible and non-contractile tail
(Adriaenssens et al., 2015). Approximately 61 % of all isolated and visualized
tailed phages belong to this morphotype (Ackermann, 2001).
Figure 1. Graphical representation of the virion structure of the tailed phages. A) Myoviridae, with
elongated head and neck whiskers (phage T4), B) Siphoviridae with flexible tail and tail fibers (phage
T5) and C) Podoviridae with icosahedral head and tail fibers (phage T7).
The third family of the order Caudovirales is the Myoviridae, whose
members have a complex and contractile tail and encode their genomes using
double-stranded DNA. The Myoviridae adhesion process to the bacterial cell
is a two-step process. Reversible adhesion occurs through interactions
between the long tail fibers of the phage with a bacterial cell receptor, which
in turn prompts a conformational change in the phage that allows for
irreversible binding to the cell through the short tail fibers (Thomassen et al.,
2003). Binding leads to a contraction of the outer tail sheath, which exposes
the inner tube, leading to penetration of the cell wall and injection of the phage
DNA into the cell. There are currently four subfamilies described in the
Myoviridae family: the Tevenvirinae, Spounavirinae, Peduovirinae and
Eucampyvirinae. These subfamilies have been classified and established
based on sequence similarity and morphological features of the contained
phages, but there are few shared characteristics that are conserved throughout
these phage subfamilies (Lavigne et al., 2009, Javed et al., 2014).
11
Phage infection
The main concern of phages is temporal: to ensure that their genes are
transcribed in the correct order so that the host cell is not lysed before progeny
virions are assembled. This process is commonly called a genetic cascade,
where each set of genes that gets transcribed during the infection opens up
another set of genes for expression, leading to a step-wise activation of each
set of genes that drives the infection. This is mediated through several
different strategies, from utilizing the host's own transcriptional machinery
with phage-encoded transcription factors, activators, repressors and antiterminators or through phage-encoded polymerases that are utilized to drive
the infection and open up the next operon for transcription (Krebs et al., 2012).
The organization of genes into functional modules is a conserved feature
among phages and can often be used to predict functions of de novo sequenced
genes through the placement of their genome (Veesler and Cambillau, 2011).
Phages can exhibit three different types of lifecycles that are dependent on
their genetic heritage: virulent, temperate or persistent (fig. 2). A virulent
phage that infects a bacterial cell progresses through the lytic cycle (fig. 2A,
left side), in which the phage genome is replicated and capsids are assembled,
followed by host cell lysis to release the progeny phages.
Figure 2. A) Phages that have the genetic prerequisites can enter both the lytic and lysogenic cycles (both
left and right cycle), while others can only follow the lytic cycle (leftmost cycle). These are called temperate
and virulent (or lytic) phages, respectively. B) Persistent infection. Host chromosome depicted in blue,
phage genome in red.
A temperate phage can coexist with a bacterial cell via the integration of its
genome either into the host or through a plasmid. When a temperate phage
infects a bacterium, there is a competition between phage expressed proteins
to either repress or initiate the lytic cycle versus the lysogenic cycle (Bertani,
1951). The outcome of the competition results in the phage entering the lytic
cycle or integrating into the bacterial genome. The prophage will be
maintained and inherited as part of the bacterial genome until a de-repression
event. While the factors that cause de-repression of the lysogenic state are
dependent on the prophage in question, DNA damage, heat shock or chemical
12
induction have been identified in phage lambda as causing excision of the
prophage and induction of the lytic pathway (Little and Michalowski, 2010).
Some phages can enter a temporary and quasi-stable state of pseudolysogeny,
sometimes called a carrier state, during cell starvation and nutrient depletion
of the bacterial cell that is being infected (Los and Wegrzyn, 2012). In this
pseudolysogenic state, the infection process will stall, providing the bacterium
more time to live and potentially a more promising environment for the phage
to propagate in the future. This pseudolysogenic state can persist (Cenens et
al., 2013a), however, even after the bacterial cell enters a high-nutrition
environment, and their genomes are asymmetrically inherited in the daughter
cells as demonstrated with the temperate phage P22 in Salmonella
typhimurium (Cenens et al., 2013b).
A third type of life style has been described in certain filamentous phages,
e.g., phage M13, in which the infection is persistent and progeny phages are
continuously released by excretion (fig. 2B). In this type of infection, the cells
remain unharmed albeit with decreased cellular growth (Chopin et al., 2002).
Phage evolution
There are several mechanisms that contribute to the evolution of phages,
such as DNA replication errors, environmental mutagens and HGTmechanisms (Abedon, 2009, Duffy et al., 2008). Single point mutations occur
at a low frequency during genome replication. These mutations are then
incorporated into progeny phages, which creates a large pool of diversity
among the phages. Due to the fast generation times of bacteria and phages,
together they constitute a good model system for insight into evolutionary
processes (Abedon, 2009). This natural plasticity of phages to adapt through
single point mutations has been demonstrated using phage T7, which uses
lipopolysaccharides (LPS) as binding receptors. The phage was subjected to
multiple passes through its wild type E. coli host and various LPS mutants,
which led to the expansion of the phage host range across these mutants
through point mutations in its tail fiber gene (Qimron et al., 2006). However,
HGT mechanisms open up more rapid evolutionary forces that operate not
only in the same bacterial species but also over species boundaries (Ochman
et al., 2000).
The integration of DNA into genomes is often facilitated through a process
termed recombination, which is responsible for the exchange or rearrangement
of genetic material. In prokaryotic cells, recombination comprises some of the
DNA repair mechanisms that maintain the integrity of the genome but also
part of the exchange of genetic material through different HGT mechanisms.
In general, two types of recombination have been described: homologous and
13
site-specific. The requirement for homologous recombination is the presence
of two similar or identical nucleotide strands, which can be joined. In E. coli,
the RecF pathway is primarily responsible for repairing single-stranded breaks
but can also repair double-stranded breaks, while the RecBCD pathway is
responsible for repairing double-stranded breaks. In general, for both
pathways, helicases are recruited to the identified DNA break to unwind the
DNA helix and nucleases degrade one of the DNA strands, which in turn
recruits the protein RecA to the free 3´ single-stranded end of the DNA. RecA
then takes part in the joining of a homologous DNA strand and a Holliday
junction is formed, followed by branch migration and strand separation after
the recombination is complete. For a comprehensive review of these
mechanisms, see references (Kowalczykowski et al., 1994, Kowalczykowski,
2000).
Site-specific recombination can be classified into two general categories:
transpositional recombination, which is further subdivided into replicative or
non-replicative transposition, or conservative site-specific recombination.
Transpositional recombination is mediated by a site-specific recombinase
protein designated transposase, which is often encoded in the mobile genetic
element. The transposase, depending on the type, can either insert the mobile
element through excision and integration, utilizing double-stranded breaks of
both the target and recipient sequences, or through replicative recombination,
in which the donor sequence is joined to the recipient, replicated and then
disjoined (Hallet and Sherratt, 1997).
Conservative site-specific recombination is a common method for
temperate phages to integrate themselves into bacterial genomes, in which
site-specific recombination proteins termed integrases mediate the insertion
of the phage genome at specific sites in the bacterial genome. Integrases are
categorized into two families: serine recombinases, which insert the DNA by
creating a double-stranded break, or tyrosine recombinases, which cleave the
DNA in two constitutive steps via single-strand breaks (Hallet and Sherratt,
1997). For reviews of these mechanisms, see references (Hallet and Sherratt,
1997, Groth and Calos, 2004).
14
Phage impact on bacterial hosts
In contrast to their lytic counterparts, temperate phages can confer
advantages via phage-encoded genes that are beneficial to the bacterial host
(Brabban et al., 2005). There are several examples in which the prophage
encodes for exotoxins, which directly promote virulence of the bacterial host
cell. For example, Vibrio cholera lysogenized by the temperate phage CTXφ
introduces cholera toxins ctxA and ctxB (McLeod et al., 2005), which cause
severe diarrhea in the host, increasing the transmission of the bacterial host
into the environment and to other hosts. There are also more discrete methods
to increase virulence gained by infecting phages, such as regulatory factors
that increase the expression of native bacterial virulence genes or phageencoded enzymes that change the expression profiles of a bacterium to
increase its virulence (Wagner and Waldor, 2002). Salmonella enterica phage
phage SopEφ encodes a type three secretion system effector protein SopE, that
promotes the invasion capability of its bacterial host (Mirold et al., 1999), and
Vibrio cholera phage CTXφ also encodes for a toxin co-regulated type IV
pilus (Karaolis et al., 1999). Other notable examples of phages that improve
bacterial host fitness include the S. enterica phage Gifsy-2, which encodes a
superoxide dismutase that enables the bacteria to survive the oxidative stress
inside phagocytes by catalyzing innate superoxide and hydrogen peroxide in
the environment into molecular oxygen and hydrogen peroxide. Another
example is the Staphylococcus aureus phage φPVL (Kaneko et al., 1998),
which encodes a human phagocyte cytotoxin that is released into the
surrounding environment and damages phagocytes (Wagner and Waldor,
2002). These examples illustrate the balance and tradeoff between the
decreased fitness of the bacteria via increased genome size by the integrated
prophage and the increased fitness conferred by the phage.
Phage–bacteria coevolution
Bacteria have numerous predators, which include phages, protozoans and
protists. It is estimated that a large portion of bacterial mortality,
approximately 50 %, is due to phage predation (Diaz-Munoz and Koskella,
2014). There are two main explanatory models to describe the complex
relationship between predator and prey, phage and bacteria. The “kill the
winner” hypothesis argues that any abundant host in the environment will
become a target for predation due to its abundance, and subsequently lead to
an increase in the predators of this prey. Thus, any successful phenotype in
the prey would eventually lead to a negative frequency selection of the
population density, where the actual benefits of a phenotype with high fitness
would be selected against when becoming abundant in the biosphere. A
15
possible parallel driving force behind the biodiversity of phages and bacteria
is the niche differentiation hypothesis, where rather than competition between
phages towards the same abundant host, a selective pressure exists to drive
differentiation towards different hosts, thereby eliminating the need for
competition and extinction of the less fit predator (Diaz-Munoz and Koskella,
2014).
Lytic phages prey on susceptible bacteria in an antagonistic manner, where
the phage goal is to produce progeny and the goal of the bacterium is to
propagate. This drives the evolution and diversification of both bacteria and
phage, and each in turn responds to an evolutionary adaptation or
circumvention of the adaptions of the other, which drives biodiversity
(Buckling and Brockhurst, 2012).
Bacterial defense systems
The close and intricate relationship between bacteria and phage is obvious
when investigating bacterial defense against phages and how phages have
counter-evolved against these defenses (Emond et al., 1998). Bacteria have
evolved strategies to counter each step of the phage infection process, from
the first step of cell adhesion to the last step in which progeny are assembled
and released into the environment. This interaction and defense against phage
predation can be divided into three discrete categories: adsorption inhibition,
restriction and abortive infections (Hyman and Abedon, 2010).
Adsorption inhibition
Adsorption prevention is the first line of defense of the bacterial cell where
the phage is prevented from attaching to and injecting its genome into the host
(fig. 3A). Phages have been shown to bind a multitude of different surface
structures of the bacterial cell wall prior to adsorption. The bacterial cell wall
component adhered to is often a vital and conserved part of the bacterial cell,
such as ferrichrome outer membrane transport protein, FhuA, which phage T5
binds to (Flayhan et al., 2012). Phage attachment to the bacterial cell is often
a two-part process, as with the Myoviridae phage T4, where the long tail fibers
interact and reversibly bind with outer membrane porin protein C (ompC) of
the bacterial cell surface, leading to a conformational change in the short tail
fibers of the phage, which irreversibly bind to the core region of the bacterial
LPS layer, leading to injection of its genome into the bacterial cell
(Thomassen et al., 2003).
16
Bacterial surface receptors
The blockage of phage receptors (fig. 3A) not only enables bacteria to be
protected against infection but can also be used by phages to prevent
superinfection in a process called superinfection exclusion (Sie). Sie has been
demonstrated with phage T5, a virulent phage that infects E. coli and expresses
a lipoprotein early during the infection process that blocks the cell receptor
that it originally adhered to, thus hindering superinfection by other T5 phages.
This masking process is also beneficial to the phage after host cell lysis and
progeny release as it stops progeny phages from adhering to lysed cell debris
(Labrie et al., 2010). An example of a competitive inhibitor produced by
bacteria is an antimicrobial protein designated J25, which binds to the iron
transporter channel FhuA in E. coli (Labrie et al., 2010). FhuA is also used by
phages T1, T5 and φ80 as an adsorption receptor with which it competes.
Antimicrobial protein J25 is produced by E. coli in low-nutrition
environments when competition for resources is high to promote its own
growth (Destoumieux-Garzon et al., 2005).
Capsule
Both E. coli and K. pneumoniae can produce an exopolysaccharide capsule
layer around the cell. This capsular layer is often considered a virulence factor
because it prevents the immune system from recognizing immunogenic
components of the bacterial cell wall, as is the case with some strains of E.
coli (Jann and Jann, 1987) and with K. pneumoniae (Podschun and Ullmann,
1998) infections. The capsule also protects the bacterium from phagocytosis.
Phages have evolved the ability to degrade these polysaccharide capsule
layers, as demonstrated by phages K1E and K1-5 (Leiman et al., 2007), which
carry virion-associated glycosidases that enable the phages to degrade and
penetrate the capsular layer to reach the cellular wall (fig. 3B).
17
Figure 3. Bacterial phage defense mechanisms. A) Adsorption inhibition, B) Restriction, C) Abortive
infection.
18
Biofilm
The ability to produce an alternative external protective barrier, or biofilm,
is a highly successful defense mechanism for bacteria (Hall-Stoodley et al.,
2004). These biofilms consists of secreted proteins, lipids, DNA and
polysaccharides that mediate the aggregation of bacteria into micro-colonies
with their own microclimates that protect the bacteria from environmental
threats such as phages and antibiotics (Bjarnsholt, 2013). Biofilms are
produced by some bacteria when they cross a threshold in terms of cell
numbers via quorum sensing mechanisms. Through the production of
biofilms, bacteria have been found to survive for prolonged amounts of time
on surfaces such as catheters, artificial implants and contact lenses, which is
problematic in health care settings (Kostakioti et al., 2013).
As with capsules, biofilms are highly efficient in masking bacteria.
Biofilms decrease the amount of active compound that reaches the bacteria
during antibiotic treatments and also prevent phage tail fibers from finding
their surface receptors on the bacterial cell (fig. 3A). Although bacterial cells
remain sensitive to an administered antibiotic, a biofilm hinders successful
antibiotic treatment, as the antibiotic is prevented from being taken up by the
bacteria and metabolized (Bjarnsholt, 2013).
Some phages have evolved a response to biofilms by encoding tailassociated biofilm degrading enzymes called depolymerases (Cornelissen et
al., 2011). These phage-associated enzymes can disrupt the protective biofilm
that shields the bacterium. Subsequently, by infecting and replicating in host
cells, phages spread their progeny further into the biofilm-shielded
microcolony and step-wise destroy the protective barrier of the biofilm.
Restriction
The second line of bacterial defense is the restriction of invading foreign
DNA. Several types of systems exist that work towards the same goal: the
digestion of foreign DNA and the protection of the host DNA (fig. 3B).
Restriction modification systems
Restriction modification (RM) systems target invading and un-methylated
DNA, while the host DNA is protected through methylation. These two
component systems are divided into different categories dependent on protein
complexity, recognition sites and their mode of action. The type II RM system
is the most disseminated of the groups and has been found in 80 % of all
sequenced bacterial genomes (Wilson and Murray, 1991).
RM systems are composed of the restriction endonuclease (REase) and the
methyltransferase (MTase), which work in concert. The REase specifically
targets and cleaves unmethylated DNA strands at specific nucleotide
19
sequences, while the MTase methylates these same nucleotides. Both proteins
exist in the cell at the same time, albeit at different concentrations, where the
REase is in abundance. Phages have evolved several different strategies to
evade digestion by RM systems, e.g., base modification to prevent
recognition, DNA masking and endonuclease blocking (Samson et al., 2013).
An example of endonuclease blocking occurs with phage T7, which co-injects
its genome with an accessory protein designated Ocr that mimics the structure
of the EcoKI REase-recognized site. Ocr has a higher affinity for the REase,
preventing digestion of the phage genome. This gives the MTase time to
methylate the phage genome, which is then protected from REase digestion,
and the infection can proceed without interruption (Atanasiu et al., 2002).
Subsequent phage progenies will have methylated genomes that will protect
them from similar RM systems until they infect a susceptible bacterial cell
that does not contain an RM system. Progeny phages from these bacterial cells
will not have methylated genomes and will be susceptible to digestion by the
same RM system.
Another escape strategy of note is the lambda-encoded antirestriction
protein Ral, which enhances the MTase activity of the infected cell (Loenen
and Murray, 1986). This changes the balance between the REase and MTase
in favor of the unprotected foreign DNA, which allows the invading and
replicating genome to be methylated and protected from REase degradation in
the cell.
CRISPR-Cas
The CRISPR-Cas system consists of genes encoding protein components,
the Cas proteins, and a nucleotide array of clustered regularly interspaced
short palindromic repeats (CRISPR). In this CRISPR array, short nucleotide
sequences called spacers are stacked between repeats. The spacers have been
acquired from previous invading foreign DNA and confer immunity against
subsequent infection of the same origin.
The CRISPR-Cas system has been denoted as an adaptive immune system
of prokaryotes (Westra et al., 2012a, Wiedenheft et al., 2012) and is a defense
system that works in two steps: an adaption/acquisition phase (Westra et al.,
2012b) in which foreign DNA is identified, cleaved, and inserted into the
CRISPR array and an interference phase (Swarts et al., 2012) in which the
acquired DNA confers immunity against a subsequent infection by a phage
with an identical sequence to the acquired DNA. In the type I CRISPR-Cas
system, the acquired DNA is transcribed and inserted into a ribonucleoprotein
complex called the Cascade complex. This Cascade complex consists of
proteins encoded by the Cas genes (casABCDE), which are assembled
together with transcribed, processed and matured RNA transcripts from the
20
CRISPR array, now called crRNA. It is this ribonucleoprotein complex that
targets specific DNA strands using its antisense RNA strand and then recruits
Cas3 for nucleolytic degradation (Yosef et al., 2012). Three major types of
CRISPR-Cas systems have been described, based on the homology of the Cas
proteins (Fineran and Charpentier, 2012).
Several evasion strategies exist for phages to overcome restriction by the
CRISPR-Cas system, for example through point mutations in the recognized
region. Another evasion strategy that has been described with Pseudomonas
aeruginosa temperate phages is a phage-encoded small protein that disrupts
the Cas and crRNA complex and thus inactivates the entire CRISPR-Cas
defense system (Bondy-Denomy et al., 2013). Interestingly, these genes are
placed in an operon that is associated with late gene expression, during which
the phage capsid genes are transcribed and translated, which is the final stage
of the genetic cascade of the infecting phage. This suggest that the antiCRISPR protein is packaged inside the virion and injected with the DNA to
immediately hinder the CRISPR acquisition process during the infection
process.
Abortive infection
Abortive infection (Abi) either drives the infected cell to arrest the phage
infection cycle or to induce bacterial autolysis. This type of phage exclusion
system has mostly been studied in Lactococcus lactis, due to its extensive use
in the dairy fermentation industry, but has also been identified in E. coli
(Chopin et al., 2005). The Abi systems characteristically allow a successful
phage infection to progresses normally until it is suddenly aborted, often just
prior to the release of progeny phages (fig. 3C). Most Abi systems that have
been identified are residents on plasmids; however, there are some exceptions
in which the Abi system is part of the bacterial genome (Samson et al., 2013).
Several types of Abi systems have been identified and characterized, and
most are dependent on a single gene for their mode of action. The targets of
the Abi systems are as diverse as the systems themselves, and hindrance of a
cell’s DNA replication process, destabilization of RNA transcripts and
arresting protein synthesis have been identified (Emond et al., 1998). Some of
these identified Abi systems are constitutively expressed in the cell, while
others are induced only after a phage infection. However, in general, all Abi
gene products are toxic for the cell when artificially induced at high levels
(Chopin et al., 2005). An example of an Abi system is the cryptic phage e14,
which is part of the Escherichia coli K12 genome. E14 encodes for the
superinfection exclusion protein Lit, which arrests all cellular translation upon
T4 infection, aborting the infection and inducing cellular suicide of the
infected cell. This cascade of events is started by the transcription of the major
21
capsid protein of T4, which interacts with the major host translation factor EFTu in its host, which in turn becomes susceptible to proteolytic cleavage by
the metalloprotease Lit. This cascade of events leads to suspension of the
translation of all proteins in the bacterial cell, and cellular death is ensured
(Bingham et al., 2000).
Another type of abortive infection of phages in bacteria is facilitated by
toxin-antitoxin (TA) systems, which are encoded on plasmids that require the
constant expression of both the toxin and the antitoxin in the host cell to ensure
its survival. Phage infection disrupts this homeostasis, and cell death follows.
In a recent study of phage infection and TA system coevolution in Erwinia
carotovora, the ToxIN system was investigated for its mode of action during
phage infection by examining escape mutants of surviving phages (Fineran et
al., 2009). These Abi escape mutant phages were subsequently sequenced, and
an identified mutated sequence was similar to the RNA antitoxin transcript of
the TA system, which mimicked the function of the original antitoxin in the
host cell, thus preventing the inhibition of phage infection (Blower et al.,
2012).
Phage therapy
The rise of antibiotic resistance
The spread of multidrug-resistant bacteria is of increasing concern to both
the medical sector and to society as a whole. Since the 1970s, only four new
classes of antibiotics have been approved, and due to the high cost associated
with the development of new antibiotics, as well as the rapid acquisition of
resistance, few new antibiotics are in the pipeline (Cooper and Shlaes, 2011).
However, over the last couple years, two new classes of antibiotics have been
approved: fidaxomicin, a narrow spectrum antibiotic that has been approved
for the treatment of Gram-positive Clostridium difficile, and bedaquiline, a
narrow spectrum antibiotic against Mycobacterium tuberculosis (Butler et al.,
2013). The acquisition and dissemination of resistance towards novel
antibiotics is a rapid process and can pass between bacterial species (Davies,
1994). For most novel types of antibiotics, resistant bacteria can be isolated
within months if not immediately after introduction. The ease of this rapid
dissemination of resistance between bacterial species is a major health
concern.
This alarming trend is visible everywhere, with a 424 % increase in
reported multi-resistant ESBL-carrying bacterial infections in Sweden during
the years 2007-2014 (Anonymous, 2013b). This type of increase is a
22
worldwide trend, though it occurs at different levels. Countries that have
generous antibiotic prescription routines or over-the-counter distribution of
antibiotics have the highest incidences of multi-resistant bacteria in the world.
Several actions have been launched from government bodies since 2004 to
combat this problem; one example is the EU-wide ban in 2006 of the use of
antibiotics in animal food, where antibiotics were added to promote growth in
livestock (Anonymous, 2005). Another action that has been taken is the
creation of the ReAct network in 2004, a global network of scientists to
promote debate and increase awareness of growing resistance against
antibiotics (Cars and Hällström, 2004). The major focus of all efforts is to
decrease the spread of multi-resistant bacteria by decreasing the usage of
antibiotics in society. This is made possible by discontinuing the use of
antibiotics for minor infections. Due to the pressing need for alternatives to
conventional antibiotics, research into bacteria- and bacteriophage-derived
antimicrobial compounds has been greatly expanded.
Traditionally, ESBL is carried on plasmids and is disseminated among the
Enterobacteriaceae family of prokaryotes, and the conferred resistance has
evolved rapidly over time. Since the isolation of the first ESBL-resistant
isolate, clinical isolates with larger resistance profiles have been
characterized, from the original characterization of resistance against
aminoglycosides in the 1970s, followed by resistance to extended spectrum
cephalosporins and to ceftazidime (Podschun and Ullmann, 1998). Resistant
clinical isolates of K. pneumoniae and E. coli have emerged against what was
previously the last-resort class of antibiotics, the carbapenems. This highlights
the importance of not only decreasing the widespread misuse of antibiotics in
today’s society but also in finding alternative treatment methods for bacterial
infections.
The history of phage therapy
Phage therapy was originally pioneered by Felix d´Herelle (D´Herelle,
1917, Schultz, 1927) as a remedy against dysentery caused by Shigella
infection during 1930s. This sparked great interest in the scientific and
medical community during that time, and many phage mixes and treatment
remedies were launched against various infections and ailments. However, the
lack of scientific understanding of phage biology, the high level of phage
specificity and the discovery of antibiotics led to the quick abandonment of
phage therapy in the West. In the following decades, only a few institutes
continued to research and use phages as a treatment method, most notably the
Eliava Institute in Georgia and the Wroclaw Institute of Experimental Therapy
in Poland (Miedzybrodzki et al., 2012). These institutes are actively involved
23
in the development and administration of phages to patients. However, the
validity of such therapies is still treated with skepticism in the West due to a
lack of adequately controlled clinical trials (Sulakvelidze et al., 2001).
Phage therapy today
Due to the high specificity and narrow host range of phages, treatment is
often more complex when compared to traditional antibiotics. A reason behind
the success of antibiotic treatment is the availability of broad spectrum
antibiotics, which only require the identification of the class of bacterium
before therapy can be administered. As both a blessing and a curse,
bacteriophage specificity requires the identification of the infecting bacterial
species before treatment can be administered. The specificity of
bacteriophages also reduces unwanted damage to the patient microflora during
therapy compared to treatment with antibiotics.
There are several commercial entities currently working within this field,
with phage products for food processing (Intralytix Inc., Micreos BV) and
human phage therapy product investigations (Ampliphi Biosciences corp.,
Novolytics ltd.). Clinical trials for phage therapy are underway; for example,
in 2009 a successful double blind, placebo and randomized phase II clinical
trial was performed using a phage cocktail consisting of six different phages.
This study investigated the safety and efficacy of using phages as a treatment
method for multi-resistant Pseudomonas aeruginosa in chronic otitis patients
(Wright et al., 2009). The treated group showed a 76 % decrease in bacterial
counts, while the placebo group showed a 9 % increase in bacterial cell density
in the place of infection.
A phage therapy study funded by Nestlé has been running extensive trials
of phage cocktails in Bangladesh (Sarker et al., 2012). Nestlé’s main
bacterium of interest is E. coli, which contaminates many sources of drinking
water, causing severe dehydration due to diarrhea in local populations. Safety
trials have been performed in adults, but the project was terminated in 2013
without notice. In 2013, the PhagoBurn project received funding from the
European Union 7th framework program to evaluate phage therapy treatment
of E. coli and P. aeruginosa infections in burn wound infections in clinical
trials (Ravat and Chatard, 2013).
There are solutions to address the narrow host ranges of phages; one is to
isolate phages with broad host ranges that utilize conserved receptors. Another
solution is to combine multiple phages with similar virulence characteristics
but with different specificities for bacterial surface receptors to produce a
phage cocktail. Ideally this cocktail would decrease the risk of bacterial
resistance developing. Bacteria acquire resistance towards bacteriophages
24
rapidly, as they do towards antibiotics, but the ease of isolation of new phages
and the low cost creates a lower threshold for investigating and replacing
phages that have become ineffective against the bacteria.
Treating untyped bacteria using bacteriophages creates novel challenges,
and strategies are to either use a pre-made phage cocktail with phages specific
against a multitude of serotypes of the same bacterial species or to tailor a
phage cocktail selection with several phages specific for the patient serotype
(Pirnay et al.). The first method requires only the isolation and typing of the
infecting species before treatment starts, while the second tailored treatment
requires not only isolation and typing of the bacteria but also susceptibility
testing of phages using an existing phage library. After susceptibility tests
have been performed, a selection of effective phages can be put into a cocktail
and administered to the patient.
Susceptibility tests to ascertain the host ranges of bacterial viruses can give
false positives or negatives for various reasons. One parameter of a phage
cocktail that must be monitored is its multiplicity of infection (MOI). This
number describes the number of phages added per bacterial cell, and thus how
many phages are expected to infect an individual bacterial cell. Problems arise
when using too high a multiplicity of infection, creating phenomena such as
lysis from without (LO) (Abedon, 2011) or lysis inhibition (LIN) (Bode,
1967). Both phenomena have been observed and investigated for phage T4,
one of the most studied phages in the Myoviridae family. LO occurs when a
bacterial cell membrane is destabilized by a multitude of phages adsorbing to
its membrane, with phage-associated and enzymatically active domains that
degrade the integrity of the cell membrane without specificity for the
bacterium (Abedon, 2011). This process leads to the premature lysis of the
host cell with no release of phage progeny.
A passive phage treatment consists of using a high MOI phage cocktail that
utilizes the ability of phages to induce lysis from without, for which a
productive infection is not needed for pathogen clearance and treatment
success. This ability of phages to non-specifically lyse bacteria can be utilized
to create passive treatment regimens, in which the outcome of the treatment is
not dependent on the successful infection and replication of the phages.
LIN has been identified and studied for phage T4 and occurs when a
successful T4 infection is underway, followed by a superinfecting T4. During
the normal progression of a T4 infection, the transmembrane T4 holin is
expressed and accumulates in the inner membrane during the late phase of
infection. It stays dormant while structural proteins are assembled and the
phage genome is packaged into the progeny capsids. At a critical
concentration, the holin oligomerizes and creates pores in the inner membrane
that allow the phage endolysin to escape the cytoplasm and degrade the outer
25
cell membrane, causing cell lysis. LIN has been demonstrated from
superinfecting T4 phages, where the protein RI interacts with the
transmembrane holin and delays oligomerization and bacterial cell lysis. If the
infected cell is challenged by a superinfection phage every 10 minutes, LIN
can be maintained indefinitely (Tran et al., 2005).
Proteins as antimicrobial compounds
Commercial interest in phage therapy is tentative because the use of natural
proteins or viruses are not patentable, and the regulatory framework for
development and testing in clinical trials creates a high threshold for academic
groups to overcome (Verbeken et al., 2012). The main difficulty in using
proteins as antimicrobial agents concerns their possible rapid inactivation
through proteolytic degradation, renal clearance or immunological blockage.
These obstacles can, however, be overcome by screening for inherently robust
and non-immunogenic proteins (Resch et al., 2011). In summary, several
frontiers in bacteria- and phage-encoded antimicrobial proteins exist; for
example, endolysins (Schmelcher et al., 2012, Walmagh et al., 2012), holins
(Ludwig et al., 2008), colicins (Hecht et al., 2012) and other protein products
have been isolated from phages (Liu et al., 2004).
Large substrate screenings are underway to identify compounds with
antimicrobial activity, both originating from phage (Liu et al., 2004) and
bacteria (Haney and Hancock, 2013). Proteins as antimicrobial compounds
have the advantage of being less immunogenic compared to whole phages,
and recombinant proteins are patentable and thus marketable.
Lysins are proteins that are expressed late in phage infections when phage
progeny have already been assembled in the infected cell. The lysins mediate
the degradation of the peptidoglycan layer of the bacterial cell wall, which
eventually leads to cellular lysis. They are effective in lysing Gram-positive
bacteria that have an exposed peptidoglycan layer, while Gram-negative
bacteria are protected due to their peptidoglycan cell wall layer being
sandwiched between an outer and inner cell membrane.
Another example of active research for bacteria-derived antimicrobial
compounds is the investigation of bacteriocins, such as colicins (Hecht et al.,
2012). Colicins are small protein molecules that can kill susceptible bacteria
by creating pores in the cell membrane or by inhibiting peptidoglycan
synthesis, thus hindering cell growth. These proteins are isolated from
bacteria, which encode for these proteins to increase their competitive
advantage in their habitat by inhibiting the growth of closely related bacterial
strains. Work is ongoing to screen, identify and characterize this class of
proteins for possible use as antimicrobial compounds (Hecht et al., 2012).
26
Recent advances in creating a hybrid bacteriocin-lysin show promise, and a
recombinant lysin has been shown to successfully target and kill Yersinia
pestis in vitro (Lukacik et al., 2012).
Enterobacteriaceae
In this thesis, we have isolated bacterial viruses targeting E. coli and K.
pneumoniae, which are both found in the bacterial Enterobacteriaceae family.
E. coli and K. pneumoniae are the most commonly isolated multi-resistant
bacterial species in the European Union; their isolation rates exceed that of
methicillin-resistant Staphylococcus aureus (MRSA) and vancomycinresistant enterococcus (VRE) (Anonymous, 2013a). Enterobacteriaceae
constitute a large family of rod-shaped, Gram-negative and facultative
anaerobic bacteria. This family of bacteria includes a wide range of bacterial
species from commensal organisms to potential human pathogens, including
E. coli, Klebsiella, Salmonella, Shigella and Y. pestis. Several bacterial species
of this family live in the intestines of warm-blooded animals, while others live
in soil or water.
Klebsiella spp.
Klebsiella is a rod shaped, non-motile, encapsulated and facultative
anaerobic Gram-negative bacterium. The genus Klebsiella contains species
that have a wide range of habitats, soil and plants, including mammalian
intestine, mucosal surfaces and skin lesions. Most Klebsiella species have the
ability to produce a thick and protective capsule around the bacterial cell,
which protects the cell from phagocytosis and other host defense mechanisms.
This capsule is composed of a diverse range of acidic polysaccharides, of
which 77 different serotypes have been described. Some of these capsular
antigens, termed K-antigens, have been associated with higher pathogenicity
in both animal models and isolated clinical samples (Simoons-Smit et al.,
1984). Serotyping of the LPS layer has revealed eight different compositions
of its outer polysaccharide layer, designated O-antigen, whereas the O1
antigen is the predominant variation in clinical isolates. This highly
immunogenic and endotoxic LPS layer is mostly concealed by the outer
capsule.
K. pneumoniae is an opportunistic and nosocomial pathogen that infects
immunocompromised patients, infants and the elderly. Patients who are
already hospitalized for other afflictions are especially vulnerable to K.
pneumonia infections, and the risk of being colonized is directly correlated
27
with duration of hospital stay (Podschun and Ullmann, 1998). While in normal
situations an infection by K. pneumoniae would be easily treatable, the
increased spread and dissemination of the ESBL resistance plasmid
throughout Enterobacteriaceae is increasing the severity and problems in
treating these infections. This increases the need for alternative treatment
methods and the need to lower the use of antibiotics in minor infections to
decrease the dissemination of antibiotic resistance in bacteria.
Escherichia coli
E. coli is a rod-shaped, Gram-negative and facultative anaerobic bacterium
that is a commensal species often found in the gastrointestinal tract of warmblooded animals. The majority of E. coli species are flagellated and are
encapsulated or produce biofilms. Due to its intimate relationship with
humans and our ability to easily cultivate this bacterium, E. coli has been one
of the most well-characterized bacterial species since its identification in
1885. E. coli is the most routinely used laboratory organism, due to its ease of
cultivation, its ability to easily accept foreign DNA and the ease of genetic
manipulation of this bacterial species. There have been described over 145
different O-antigens and over 80 different K-antigens in E. coli species (Davis
et al., 1973). The majority of E. coli species are commensal and assist the host
with the breakdown of food and production of vitamin K (Bentley and
Meganathan, 1982), but several pathogenic serotypes have been described that
cause diarrheal diseases or colonize extra-intestinal sites, such as the urinary
tract (Lai et al., 2013).
28
Aims
The purpose of this thesis project has been to isolate, purify, and
characterize phages and to test different hypotheses regarding phage therapy
efficacy, in vitro and in vivo, for single phages and on phage mixtures. The
long term goal is to establish a phage collection and strategies for curing multiresistant Klebsiella pneumoniae infections in humans.
These aims have been realized in the papers of this thesis as follows:

Isolation and characterization of phages against multi-resistant
Klebsiella pneumoniae and Escherichia coli (Paper I and Paper II)

Characterization and annotation of phage genomes to identify
possible toxins (Paper I, II)

Creation of novel phage cocktails and test them against a selection
of Klebsiella pneumoniae clinical isolates (Paper III)

Testing treatment efficacies in vitro and in vivo (Paper IV)
29
Results and discussion
Paper I
A suggested new bacteriophage genus, “Kp34likevirus”, within the
Autographivirinae subfamily of Podoviridae
Paper I is a product of our continuous work to isolate and sequence novel
phages from waste water effluent plants in Stockholm, Sweden. The decreased
cost of whole genome sequencing has resulted in an abundance of phage
genomes being submitted to NCBI GenBank, but there is limited work being
done on the taxonomical classification of these phages. Newly sequenced
phages are often loosely associated into large super-genera based on similarity
to the closest available phage genome, as with several recent cases with
Phikmvlikevirus (Drulis-Kawa et al., 2011, Lin et al., 2014).
There are some ongoing efforts to classify and categorize phages into new
families and genera, for example with Podoviridae (Lavigne et al., 2008),
Myoviridae (Lavigne et al., 2009) and Siphoviridae (Adriaenssens et al.,
2014). Within the Podoviridae subfamily of Autographivirinae there are
currently three established genera: the T7likevirus, Sp6likevirus and
Phikmvlikevirus (Lavigne et al., 2008). The results of this study increased the
taxonomical resolution of the Autographivirinae subfamily of phages. Based
on sequence homology to Klebsiella phages KP34 (Drulis-Kawa et al., 2011),
NTUH-K2044-K1-1 (Lin et al., 2014), and F19 (KF765493), newly isolated
phages SU503 and SU552A were characterized and compared to each other
and the Phikmvlikevirus genus. These five phages demonstrate a high
nucleotide pairwise identity to each other, between 75.1 – 75.9 %, with 29
conserved genes between themselves and similar gene arrangements. In
comparison to members of the taxonomically close Phikmvlikevirus, 18
conserved genes are shared across the genus boundaries. Within the subfamily
of Autographivirinae, a total of nine genes are conserved between the type
phages of the genera T7likevirus, Sp6likevirus, Phikmvlikevirus and the
proposed “Kp34likevirus”. Furthermore, the phages of the proposed phage
genus “Kp34likevirus” show distinct differences compared to the phages in
the closest relative phage genus, the Phikmvlikevirus. The setup and gene
variants of the lysis cassette genes differ between the genera: the KP34-like
30
phages have a unimolecular spanin (u-spanin) and a spanin-holin-endolysin
setup, while the φKMV lysis cassette has the gene arrangement holinendolysin-spanin with a dual protein spanin (i-spanin/o-spanin) complex
similar to that of phage lambda.
The Autographivirinae obligatory RNA polymerase also shows divergence
between the KP34-like phages and the Phikmvlikevirus group members. When
comparing RNA polymerase specificity and recognition loops, they are highly
conserved between the member phages of the proposed “Kp34likevirus” and
divergent from the phages in the Phikmlikevirus genus group. Among the
KP34-like phages, the recognition loops are identical, while the specificity
loops show a minor divergence of four amino acids out of 25, just after the
DNA interacting basepairs of the loop. The conservation of the DNA
interacting regions of the RNA polymerase suggests the use of a genusspecific promoter sequence within the proposed “Kp34likevirus” phages. This
suggests that a conserved genus of phages that is widespread in the biosphere
has been identified.
31
Paper II
Genomic, Proteomic, Morphological, and Phylogenetic Analyses of
vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology
Paper II describes vB_EcoP_SU10 (SU10) as an E. coli phage with the rare
C3 morphology that has an elongated head with a length-to-width ratio of over
2.5. Phages with this rare phenotype have only been fully described in a few
cases (Ackermann, 2001). Morphological analysis using transmission electron
microscopy (TEM), ultra-thin section electron microscopy, and scanning
electron microscopy (SEM) was performed on the phage and on infected
bacterial cultures at different time points in the infection cycle. Thin section
micrographs showed that phage capsids are arranged in a honeycomb-like
structure before and during the DNA packaging process in the host cell. No
relationship between the genome size and head length was observed in
comparison to φEco32 (Pavlova et al., 2012), Serratia marcenses phage
KSP100 (Matsushita et al., 2009), Salmonella enterica phage 7-11 (Kropinski
et al., 2011) and E. coli phage NJ01 (Li et al., 2012), all of which are C3
morphology Podoviridae phages.
Comparison of the tail lengths between free and adsorbed virions revealed
more than a two-fold difference in length. Two possible explanations exist for
this observation: either there is a post-adsorption change in the virion tail
structure or the observed difference is an artifact of the two different
preparation methods for the ultra-thin sectioning TEM and regular TEM.
Genome analysis and annotation of SU10 revealed over 40 % nucleotide
sequence similarity and also a gene arrangement in common with phage
φEco32 (Pavlova et al., 2012). Based on these similarities, a phage-encoded
sigma factor and a sigma-70 inhibitor were identified.
Transcriptional in silico analysis suggested that several structural genes are
transcribed early in the infection process through host sigma-70 promoters.
Ultra-thin sectioning micrographs confirm these structural proteins as early as
five minutes after adsorption to host cells.
Structural proteins were investigated using mass spectrometry (MS) from
mature and PEG-precipitated virions, where 34 proteins out of 125 predicted
coding sequences were identified and confirmed. The phages contain a
ribosomal slippage site that can create an alternative major capsid gene
variant, which has also been confirmed through MS.
Phylogenetic analysis was performed with C1 and C3 Podoviridae phages
that demonstrated sequence similarity with the major capsid and scaffolding
protein of SU10 to investigate the evolutionary divergence of the elongated
head morphology. Based on these phylogenetic analysis, the C1 and C3
morphology phages diverged approximately 280 million years ago and
32
adapted to different bacterial family hosts. The C3 morphology phages infect
members of the Enterobacteriaceae family, while the C1 morphology phages
infect members of Pseudomonadaceae bacteria.
Paper III
A novel phage cocktail inhibiting the growth of 99 β-lactamase-carrying
Klebsiella pneumoniae clinical isolates in vitro
The antibiotic resistance of Enterobacteriaceae is often conferred through
a plasmid-mediated enzyme that has the ability to hydrolyze and inactivate βlactam antibiotics, which includes penicillins and third-generation
cephalosporins (Doern, 1995). Additional resistance to carbapenem
antibiotics (ESBLcarba) has emerged (Jacoby, 1997), further reducing the
number of effective treatments.
A phage cocktail consisting of six phages that target K. pneumoniae isolates
was assembled from a collection of 60 isolated phages. The phages were
selected for their ability to prey on 24 extended spectrum β-lactamase (ESBL)carrying K. pneumoniae isolates. The cocktail was then assessed against a total
of 125 K. pneumoniae isolates with different resistance profiles, including
carbapenem-hydrolyzing ESBLcarba isolates. As part of this study, host range
screenings of the phage cocktail and the individual phages were performed
using standard materials and methods adapted for antibiotic resistance
screenings in clinical laboratories to determine the ease of adaptation of phage
screenings to these labs. The use of standard protocols and equipment only
required slight adjustments to determine phage susceptibility in clinical
laboratories.
The phage cocktail inhibited the growth of 99 of the 125 (79 %) clinical
isolates. However, two phages out of the six were responsible for the majority
of the bacterial inhibition across the isolates, and productive infection was not
confirmed to be the cause of the bacterial growth inhibition.
33
Paper IV
Evolution of virulence in Klebsiella pneumoniae treated with phage
cocktails
Paper IV investigates the impact of phage predation on bacterial virulence.
While resistance is a major problem with traditional antibiotics, the short
generation times and ongoing co-evolutionary battle between bacteria and
phage have created a large natural reservoir of phages that can be used to
overcome bacterial phage resistance. The assembly of phage cocktails that
adsorb to different bacterial receptors decreases the risk of resistance
developing during treatment (Levin and Bull, 2004, Chan and Abedon, 2012).
Selecting phages that utilize conserved bacterial receptors that are also
virulence factors has previously been shown to attenuate the pathogenicity of
phage-resistant bacteria (Filippov et al., 2011, Laanto et al., 2012).
The study sought to isolate phage-resistant bacterial clones that had grown
resistant to one or more phages and to characterize these bacterial clones in
terms of growth characteristics, biofilm production and virulence in a
eukaryotic host. K. pneumoniae isolate 07RAFM-KPN-524 (CTX-M9 ESBLharboring) was isolated from a patient in Sweden in 2007 and chosen as a
routine bacterial host. Four strictly lytic phages that produced halo plaques
were isolated for this study, suggesting the presence of biofilm-degrading
depolymerases (Hughes et al., 1998).
In order to obtain phage-resistant mutants, several microcosms were set up
with combinations of the four phages to facilitate predation on 07RAFMKPN-524 continuously for one week. Phage-resistant bacteria were isolated,
growth rate characteristics and biofilm production determined for each of the
bacterial clones. Clones were then used to infect Galleria mellonella, a model
organism for virulence testing in eukaryote infections (McLaughlin et al.,
2014). Three out of the four treatments that contained a single phage showed
an increased bacterial growth rate (phages B, H and K; p = < 0.001), while a
single multi-phage combination (BHX) showed a decrease in maximum
growth rate compared to an untreated control (p < 0.001).
Biofilm production was lowered in the phage resistant bacteria from multi
phage treatments compared to the single phage treatments (p < 0.001).
Individually, two phages decreased biofilm production (phages K and X) in
comparison to the untreated control (p = 0.001 and p = 0.002).
While these results were surprising, and our hypothesis was that phage
predation would decrease the virulence of the bacteria in the in vivo model, it
has been previously reported that increased growth rate could be a plastic
response to phage predation in the bacteria. These results suggest that terminal
34
investment (Poisot, 2012) plays some role in the increased growth rates
observed; however, further investigation is required.
Concluding remarks and future perspectives
The aim of this thesis has been to gain a better understanding and increase
our knowledge of lytic bacteriophages that infect and kill multi-resistant
bacteria and to sequence, annotate and characterize these phages. One goal
has also been to test phage cocktails against a selection of multi-resistant
bacteria. We have isolated bacteriophages against ESBL-carrying
Enterobacteriaceae with a special focus on K. pneumoniae. The long term
goal has been to create a phage cocktail that is effective against a range of K.
pneumoniae isolates that would effectively lyse a large selection of clinical
isolates.
Two phages that were isolated during the study exhibited large sequence
similarities to each other and also to already-sequenced Klebsiella phages
KP34, F19 and NTUH-K2044-K1-1. From our analysis, we suggest the
creation of a new Podoviridae genus called “Kp34likevirus” within the
Autographivirinae subfamily (Paper I). However, in contrast to the member
phages of the proposed “Kp34likevirus” genus, the existing Phikmvlikevirus
member phages φKMV, LKA1, LIMElight and LIMEzero have demonstrated
distinct differences between themselves, and their relationships to each other
should be re-examined (Adriaenssens et al., 2011, Ceyssens et al., 2006).
Based on phylogenetic analysis of the conserved genes of the phages that were
compared in this study, the monophyletic clade of Pseudomonas phages
φKMV, PT2, PT5, MPK6, MPK7, LUZ19, LKD16 and φkF77 comprises
candidates for a study to revise the Phikmvlikevirus genus.
In paper II, we characterized the polyvalent Podoviridae phage
vB_KpnP_SU10 (SU10) with the rare C3 morphology, a phage that infects a
broad range of the E. coli reference collection (ECOR) (Ochman and Selander,
1984) but also two clinical ESBL-harboring E. coli isolates. From our study,
we suspect that the process of phage infection and assembly are not as
straightforward as first thought. SU10 has both host and phage promoters
upstream of some structural genes, for example the capsid gene, scaffold gene
and portal protein gene. The classical model of a phage infection process can
be divided into three parts: 1) transcription of early phage genes and
commandeering of the host transcriptional apparatus, 2) transcription of
middle genes and replication of the phage genome and 3) transcription of late
genes, e.g., structural genes and cell lysis. This set of events suggest that DNA
packaging is one of the last steps of the infection cycle; however, with
35
observations from our in silico analysis of transcriptional starts combined with
ultra-thin TEM micrographs, we observed the assembly of capsids early in the
infection process.
Further studies of the head elongation process and the mechanisms
underlying size determination of the elongated head morphology are needed
to determine if, for example, the scaffolding protein or a unidentified tape
measure protein is responsible for the capsid length.
In paper III, new phages were isolated and assembled into an experimental
phage cocktail, which was assessed for in vitro activity against 125 clinical
ESBL-harboring K. pneumoniae isolates. These isolates were collected
worldwide and included different types of ESBL-resistant strains, including
metallo-β-lactamase ESBLcarba resistant strains. This pilot study was to
determine the feasibility of a pre-made phage therapy cocktail against a wide
selection of unknown isolates. Phages that possessed good growth
characteristics and a wide host range were selected and assembled into a phage
cocktail. Further characterization of the phages in the cocktail is
recommended, such as their average burst size, latent period and adsorption
rate. When compared, these characteristics could improve the selection of
phages for future cocktails. By adding or replacing phages in the current
cocktail, we can extend the host range to include, for example, capsular
serotype K2. Optimization can be performed by both identifying the epitope
that the phage receptor recognizes and by selecting phages that use essential
surface proteins as receptors, thus decreasing the probability of resistance by
receptor mutation/variation in the bacteria. However, to ascertain the
receptors that phages use for adsorption, deletion mutants must be constructed
in a susceptible host or through whole genome sequencing of the wild type
bacterial host and subsequent phage resistant strains.
The phage cocktail could next be evaluated in pre-clinical in vivo trials
using mice in a lung infection model (Debarbieux et al., 2010). However, most
animal models do have drawbacks that must be considered, as the infecting
bacterial isolate must be optimized for disease progression in the animal. This
optimization consists of selecting pathogenic strains of the bacterium that
result in quick disease progression and 100 % lethality. Another drawback of
current phage therapy studies is that phage inoculation occurs immediately
prior to or after the administration of pathogenic bacteria, and thus these
results should be viewed with some caution. These protocols should be
adjusted for more realistic disease progression, i.e., with a slow infection and
no phage treatment until symptoms arise that would simulate a real-world
infection scenario.
Another aspect of phage therapy that must be addressed is the current
legislation that regulates medicinal products, which has been written with
36
traditional antibiotics and medicinal products in mind, and future phage
therapy products and their standing in these regulations must be investigated
(Verbeken et al., 2014). Furthermore, the possibility of using engineered
phages must be addressed, both legally and biologically. The template for this
engineered phage would be a fully characterized, sequenced and annotated
phage, selected for its high burst size and short latency period. By replacing
the recognition region of the tail fibers, one could switch the specificity of the
phage and thus create a fast track for pharmaceutical use, lessening the
legislative burden for phage therapy. By using one model phage for each
bacterial genus, co-evolutionary aspects of the phage bacteria could be
exploited, such as promoter and codon usage adaption. The aspect of
acceptance from society in using genetically modified bacterial viruses as
therapeutic agents must also be considered before their use as medicinal
products.
Paper IV explores the relationship between phages and bacteria and the
effects that phage predation has on the bacterium, whether it be with a single
phage or several phages simultaneously. Interestingly, we discovered that the
selection of the phage used in therapy is important, some of the resulting
phage-resistant bacteria in our study exhibited an increased growth rate and
lethality to the eukaryotic host. To further understand the mechanism
underlying the increased virulence effects in the bacterium, further
characterization of phages as well as phage-bacteria interactions should be
performed. This is in contrast to studies that have shown attenuated bacterial
virulence in phage-resistant bacteria from phages that adsorb using bacterial
receptors that also serve as virulence factors.
Phages H and X in the study (Paper IV) demonstrate two opposite effects
on the host bacterium, and their difference in gene content, receptor usage and
growth characteristics must be investigated to understand and predict which
phages are suitable for phage therapy.
37
Acknowledgments
First and foremost I would like to thank my wife Joanna and my family for
the support and encouragement over these years. I would also like to thank my
supervisor Dr. Anders S. Nilsson for taking me in as PhD student, and my cosupervisor Professor emerita Elisabeth Haggård-Ljungquist for all her
suggestions, help and support during these years.
For making my time at Stockholm University more enjoyable, I would like
to especially thank Jaroslav Belotserkovsky, Sidney Carter and Bo Lindberg.
Good friends and great colleagues!
I would also like to mention and thank previous members of the group:
Lina Sylwan, Hanna Nilsson, Richard Odegrip, Sridhar Mandali, Marios
Hellstrand, David Berta and Andreas Bertilsson. It wouldn’t have been the
same without you! From the corridor, with shared moments and lunches
together: Asal, Alicia, Ali, Fredrik, Kun, Mattias, Melanie, Mohea, Noushin
and Sara.
For bringing new perspectives to old problems, sharing a lab bench and
projects together, Anni-Maria Örmälä-Odegrip. Your refreshing energy,
optimism and “can do” attitude really made my last year go so much faster!
Many thanks to my international collaborations Andrew Kropinski,
Zuzanna Drulis-Kawa and her group for the joint work with paper I. Thank
you for the great teamwork and collaboration! For assistance with the thesis
preparation I would like to mention Asem Abd El Monem Fatom (figure 1),
Susanna Hua (figure 3), Callum Cooper and American Journal Experts for
editing and proof-reading the thesis.
38
For making my days at Stockholm University more fun and engaging, I
would like to thank the early morning gym group at the SU staff gym: Peter
Reinhed and Wilhelm Widmark for all those mornings together, the afternoon
coffee club: Ainars Bajinskis and Sara Shakeri Manesh for all the nice talks,
shared moments and coffee together, and the technical staff for all the help I
received from Inga-Britt Olausson, Görel Lindberg, Eva Eyton and Eva
Pettersson during these years.
My grateful and cordial thanks go to Stiftelsen Olle Engquist Byggmästare,
for their funding of the project over the years and to the foundation Sven and
Lilly Lawskis fond for their stipend and travelling grants during my first two
years.
39
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