Virology 398 (2010) 7986

Contents lists available at ScienceDirect

Virology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o

A tale of tails: Sialidase is key to success in a model of phage therapy against

K1-capsulated Escherichia coli
J.J. Bull a,c,d, E.R. Vimr e, I.J. Molineux b,c,
a

Section of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA
Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX 78712, USA
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA
d
Center for Computational Biology and Bioinformatics, The University of Texas at Austin, Austin, TX 78712, USA
e
Laboratory of Sialobiology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA
b
c

a r t i c l e

i n f o

Article history:
Received 19 September 2009
Returned to author for revision
16 October 2009
Accepted 23 November 2009
Available online 16 December 2009
Keywords:
Bacteriophage
K1 capsules
Sialic acid
Phage therapy

a b s t r a c t
Prior studies treating mice infected with Escherichia coli O18:K1:H7 observed that phages requiring the K1
capsule for infection (K1-dep) were superior to capsule-independent (K1-ind) phages. We show that three
K1-ind phages all have low tness when grown on cells in serum whereas tnesses of four K1-dep phages
were high. The difference is serum-specic, as tnesses in broth overlapped. Sialidase activity was associated
with all K1-dep virions tested but no K1-ind virions, a phenotype supported by sequence analyses. Adding
endosialidase to cells infected with K1-ind phage increased tness in serum by enhancing productive
infection after adsorption. We propose that virion sialidase activity is the primary determinant of high tness
on cells grown in serum, and thus in a mammalian host. Although the benet of sialidase is specic to K1capsulated bacteria, this study may provide a scientic rationale for selecting phages for therapeutic use in
many systemic infections.
2009 Elsevier Inc. All rights reserved.

Introduction
The use of phages to treat bacterial infections, phage therapy, is an
enigma of Western medicine. Proposed and practiced before the
development of antibiotics and before phage genetics was even
understood, phage therapy was disgraced and displaced in the West
by antibiotics during the 1940s, although it survives even today in
Eastern Europe. Only now, amid an ever-growing threat from
antibiotic resistant bacterial pathogens, is it being entertained again
in the West. Yet after more than a decade of promulgation in the U.S.,
it has been approved only for treatment of processed food and plants
and has seen little interest from Big Pharma ( Brussow, 2005; Carlton
et al., 2005; Coates and Hu, 2007; Hanlon, 2007; Leverentz et al., 2003;
Mattey and Spencer, 2008; Projan, 2004; Schoolnik et al., 2004;
Soothill et al., 2004; Sulakvelidze, 2005).
In concept, phage therapy seems invincible: a self-replicating
organism selectively killing only a pathogen, amplifying itself only
where it is most neededsites where the pathogen is lurking and
abundant. Yet successful treatment with phages is necessarily
founded on dynamic principles just as pharmacodynamic properties
determine whether a drug that kills a bacterium in vitro will actually
be effective in vivo (Bull and Regoes, 2006; Levin and Bull, 1996, 2004;
Corresponding author. Section of Molecular Genetics and Microbiology, The
University of Texas at Austin, Austin, TX 78712, USA.
E-mail address: [email protected] (I.J. Molineux).
0042-6822/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2009.11.040

Payne and Jansen, 2001, 2003). A phage that does not amplify fast
enough or cannot access its host in some tissues may fail clinically,
even if it works well in the lab. Most lab practitioners are aware that
phages do not sterilize cultures of cells even under ideal conditions
and may not even kill cells under poor growth conditions, but this and
other empirical realities have not helped balance the promise with the
prospects of a viable phage therapy enterprise.
Much experimental and empirical research on phage therapy has
omitted consideration of dynamics, relying on qualitative outcomes.
However, a systematic study using Escherichia coli and laboratory
phages in a mouse model has been initiated (Denou et al., 2009; Weiss
et al., 2009). Nevertheless, there is yet little work to provide
mechanistic insight on how to improve treatment, short of haphazardly testing multiple phages. The use of genetically engineered
phages has shown that rational design of phage properties can
improve efcacy (Hagens and Blasi, 2003; Hagens et al., 2004; Lu and
Collins, 2007; Westwater et al., 2003), and articial selection of a
phage in vivo led to a reduced clearance rate of phage in mice (Merril
et al., 1996). So some progress is being made toward understanding
how to improve phage therapy success.
One promising system for discovering the bases of phage therapy
success is a mouse infection model developed over 25 years ago
(Smith and Huggins, 1982). Inoculation of 5 107 bacteria (E. coli O18:
K1:H7) into the leg of a mouse was nearly always fatal after 24 h
unless treated early by phages or antibiotics. The phages were wild
isolates from sewage and pig farms in England. However, in this

80

J.J. Bull et al. / Virology 398 (2010) 7986

Table 1
Notation and origins for the seven phages characterized in detail.
Namea

Family

Genome
size (bp)

Origin/identity

K1-dep(1)

Siphoviridae

41,632

K1-dep(2)

Podoviridae

45,251

K1-dep(3)
K1-dep(4)

Podoviridae
Siphoviridae

44,385
43,587

K1-ind(1)

Siphoviridae

42,292

K1-ind(2)

Siphoviridae

42,765

K1-ind(3)

Siphoviridae

43,461

Atlanta, GA sewage
(LH in Bull et al., 2002).
Formally named K1H
K1E (Gross et al., 1977;
Stummeyer et al., 2006;
Tomlinson and Taylor, 1985)
K1-5 (Scholl et al., 2001; 2004)
Superior growth rate in competition
of 2,000 K1-ind and K1-dep phages
from Austin, TX sewage, subjected
to serial passage in serum on CAB1.
Formally named K1G
Atlanta, GA sewage
(LW in Bull et al., 2002)
Formally named K1ind1
Superior growth rate of 54 K1-ind phages;
from Austin, TX and Moscow, ID, grown
in serum on CAB1. Formally named K1ind2
Superior growth rate in competition of
450 K1-ind phages from Austin,
TX sewage, subjected to serial passage
in broth on a K1E-resistant CAB1.
Formally named K1ind3

a
K1-dep indicates that the phage requires the K1 capsule, K1-ind that the phage does
not require K1.

heterogeneous collection of phages, some performed much better

than others, achieving near 100% survival when administered
simultaneously with, but separately from, the bacteria. Good phages
were of a type that required the bacterial K1 antigen for infection (K1dependent, or K1-dep phages); of 9 isolates, 8 rescued 10 of 10 mice,
and the 9th rescued 9 of 10. Six apparently independent phage
isolates that did not require the K1 antigen were tested (K1independent, or K1-ind phages), rescuing on average 33% of mice,
the best rescuing 60%.
No further investigation was undertaken by Smith and Huggins
of the mechanisms underlying the difference between the two types
of phages, except for a casual note that K1-ind phages were poor at
lysing cultures in broth and formed smaller plaques than K1-dep
phages. The authors further argued that the success of K1-dep
phages lay in their ability to rapidly destroy the bacteria, a
perspective that inspired, and was supported by, several theoretical
studies (Levin and Bull, 1996, 2004; Payne and Jansen, 2001, 2003).
These observations raised the specter that K1-ind phages grew
poorly under many conditions and might have a genomic property
incompatible with fast growth (cf. Bull et al., 2004), and in turn,
that poor growth rate was inimical to treatment success. Bull et al.
(2002) compared single K1-dep and K1-ind phage isolates using a
different E. coli O18:K1:H7 strain, and obtained quantitatively
similar results to those reported by Smith and Huggins (1982). In
addition, growth rates (absolute tness) of both phages were
formally quantied on cells grown in broth or serum. The K1-dep
and K1-ind phage grew equally well in broth but the K1-ind phage
performed far more poorly in serum although there was considerable variance between replicate assays.
If the different in vivo performance of K1-ind and K1-dep phages is
general, not merely an accident of the phages sampled, the system
could offer insight to the determinants of phage therapy success.
Understanding the cause of the difference could lead to a rational a
priori choice of phages that perform well in treatment. The goal of the
present study is to resolve the basis of the difference in tness of K1ind and K1-dep phages, extending the sample to a larger set of K1-ind
phages in search of some with high in vivo tness. Following the
precedent of Bull et al. (2002), the in vivo environment is represented
by using serum as growth medium.

Results
Phage samples
The phages subjected to detailed assays are described in Table 1,
each capable of growing on the host CAB1 (E. coli O18:K1:H7). For
ease of presentation, they will be denoted K1-ind(i) and K1-dep(j), to
facilitate distinguishing whether they require K1 for infection, with
subscripts distinguishing their identities within a type. Four phages
were chosen without a priori knowledge of or bias toward tness in
broth or serum, only that they formed plaques on LB agar plates with
CAB1 as host. The others were chosen for high growth rates from
moderately large samples of otherwise uncharacterized phages.
K1-dep phage tness is superior, but only in serum
Prior work raised the possibility that K1-dep phages have higher
tness than K1-ind phages in the environment of a mammalian host
but sampling was limited and assays were variable. Here we consider
the generality of that model. As before, the growth rate of a phage
(measured as doublings per hour) describes its ability to amplify its
numbers when hosts are not limiting, and it is used here as a measure
of phage tness. For example, a growth rate of 10 doublings/
h indicates a 1024-fold (210) increase in numbers each hour, whereas
a tness of 15 indicates a 32,768-fold increase (under the assay
conditions). A phage's growth rate reects its ability to rapidly expand
during treatment and kill bacteria to the extent that growth rate
measured in vitro mirrors growth rate in vivo. The assays here used
either broth or bovine serum, the latter interpreted as more closely
approximating a within-host environment.
Growth rates of the seven phages were measured on CAB1 (Fig. 1).
In broth, there was considerable overlap between the K1-ind and K1dep groups. There was a clear separation between the two groups in
serum: all K1-dep phages grew faster than all K1-ind phages. Yet
there was substantial variance within each group; the best K1-dep
phage exceeded the next-best K1-dep phage by the same amount as
the difference between the worst K1-dep phage and the best K1-ind
phage (4.2 doublings/h). The K1-dep phage isolated from competition
in serum had the highest growth of all other phages in serum but not
in broth. Likewise, the K1-ind phages isolated from competitions had
higher growth rates than other K1-ind phages in the competition
environment but not in the other environment. Thus, while there may
be a correlation between tnesses in different environments, within
each phage type, the correlation is not perfect.
Common to most phages is that tness is higher on cells grown in
broth than in serum. The suppressive effect of serum is merely far
greater for the K1-ind phages. The general effect of serum in reducing

Fig. 1. Fitnesses (doublings/h) of four K1-dep phages and of three K1-ind phages
measured in LB (broth) and in bovine serum. Points give means, bars are 1 standard
error. The diagonal line indicates equal tness in broth and serum.

J.J. Bull et al. / Virology 398 (2010) 7986

81

tness of both phage types may have a different cause than its
differential effect on K1-ind phages. Bacterial physiology may simply
be less conducive to supporting phage growth in serum than in broth.
Adaptation of a K1-ind phage fails to overcome the tness constraint
Given that we could not isolate K1-ind phages with high tness in
serum, we attempted to evolve one. Both phages and bacteria
commonly show tness improvement when adapted to growth
conditions (e.g., Bull and Molineux, 2008; Ebert, 1998; Fong et al.,
2006). However, 50 h of serial passage of K1-ind(1) in serum failed to
elicit an increase in its tness (data not shown, the net population
expansion was approximately 10300). Thus the limitation of K1-ind
phages to have low tness on cells grown in serum appears to be
strong.
K1-ind and K1-dep phages do not have fundamentally different genomes
A possible explanation for the different serum tnesses of K1-dep
and K1-ind phages is that the two phages represent fundamentally
different genomic types, each with different limitations in their
abilities to grow in some environments. For example, phages of the
Podoviridae exhibited higher evolved tnesses than phages of some
other families given similar opportunities for adaptation (Bull et al.,
2004). The two previously characterized phages in our sample of
seven are Podoviridae, and both are K1-dep (Table 1).
To explore phage identities, genome sequences were determined
for the 5 remaining phages, representing two K1-dep and three K1ind isolates obtained from sewage (annotations provided in Table
S1). Surprisingly, all ve phages are extremely similar in genome
structure, even though K1-dep(1) and K1-ind(1) were isolated
10 years before the other three and from a distinct geographical
area. (Note that phages isolated from Austin sewage were not
amplied on CAB1 before isolation.) The ve genomes range from
41632 bp to 43857 bp and with one major exception, contain the
same complement of genes. Gene order is essentially syntenous,
differing only by minor indels, and in the cases of K1-dep(4) and
K1-ind(3), by the acquisition of, respectively, a putative homing
endonuclease and an uncharacterized Listonella phage protein. A
dotplot analysis of four genomes reveals stunning similarity, even
between K1-dep and K1-ind types (Fig. 2; K1-ind(3) is not included
as it is virtually identical to K1-ind(2), with 98% nucleotide identity
over the entire genome). Their closest relatives in the database, as
judged by BlastP searches of predicted orfs are the Salmonella
typing phages SETP3 (De Lappe et al., 2009) and KS5, although
nucleotide similarity to putative homologs is not high. It is
noteworthy, if not surprising, that the ve phages isolated for this
and earlier work, although similar among themselves, differ
profoundly from the two K1-dep phages that were included in
our sample based on a priori knowledge of their ability to infect K1capsulated bacteria.
Only K1-dep phages possess endosialidase activity
The major difference among the ve genome sequences lies in
the putative tailspike gene. Both K1-dep phage tailspike genes
contain an endosialidase, with greater than 96% amino acid
sequence identity to the enzyme characterized from the tail of
phage 63D (Kataoka et al., 2006; Machida et al., 2000a; Machida et
al., 2000b). Both K1-dep(2) and K1-dep(3) (K1E and K1-5,
respectively) also have documented endosialidases (Leggate et al.,
2002; Leiman et al., 2007; Muhlenhoff et al., 2003). In contrast,
sequences of the three K1-ind phages indicate a glycosidase that is
most closely related to the coliphage HK620 tailspike. The HK620
enzyme is an endo-N-acetylglucosaminidase that degrades the Oantigen of E. coli O-18A1 serotype (Barbirz et al., 2008).

Fig. 2. Dot plots of the phages K1-ind(1), K1-ind(2), K1-dep(1) and K1-dep(4). The
main difference between K1-dep and K1-ind types is in the tailspike gene (arrows).
Window size is 19 bases, and each dot implies identity over all 19 bases. K1-ind(3) is
not included because it is virtually identical to K1-ind(2).

Although sequence similarity between K1-dep and K1-ind phages

is low across these tailspike enzymatic domains, it is high on both
anks. Between the K1-dep and K1-ind phages, the N-terminal 3546
amino acids of the tailspike are 100% identical, and the N-terminal
115, likely the tail-binding domain, are 78% identical. (The three K1ind phages contain identical 5-terminal nucleotide sequences of the
tailspike gene.) A region of N100 bp identity lies upstream of the
tailspike initiation codon in all ve phages, and this region is extended
upstream at 95% similarity for 3.5 kb. Downstream of the tailspike
gene, high similarity between the K1-ind and K1-dep phages again
becomes apparent, also persisting for several kb. These closely related
K1-ind and K1-dep phages thus present a clear example where an
enzyme domain has been exchanged by recombination while
maintaining a common tail-binding domain of the tailspike (Barbirz
et al., 2008; Leiman and Molineux, 2008; Scholl et al., 2004;
Stummeyer et al., 2006).
Inference of sialidase activity in all K1-dep phages and its
absence in all K1-ind phages was conrmed by direct enzyme
assay of virions. All K1-dep phage virions contain sialidase,
whereas the enzyme is absent in all K1-ind virions (data not
shown). From the combined sequence comparisons and enzyme
assays, the essential difference between K1-dep and K1-ind phages
lies in their tailspike, with K1-dep phages having an enzyme that
degrades the bacterial K1 capsule and K1-ind phages having an
enzyme that degrades O-antigen.
Exogenous endo-N restores most of a K1-ind phage's tness in serum
The invariant absence of sialidase activity with K1-ind virions
raises the possibility that the K1 capsule of serum-grown cells is
responsible for the major tness decit of K1-ind phages. One test of
this model is provided by the addition of free endo-N-acylneuraminidase (endo-N) to the culture. If the lack of sialidase in K1-ind phages
is responsible for low tness in serum, and if the capsule is more of a
barrier in serum than in broth, addition of exogenous endo-N should
greatly increase tness in serum but have little effect in broth. The
consequence of endo-N addition in serum was a tness increase of

82

J.J. Bull et al. / Virology 398 (2010) 7986

medium, were not signicantly higher with addition of endo-N [t(6)

= 0.9, P 0.2, one-tailed t-test].
In contrast, genome internalization and phage development
appeared to be greatly enhanced by the addition of endo-N. Following
adsorption of K1-ind(1) to CAB1 in serum, addition of endo-N resulted
in a 15-fold increase in burst size (Fig. 4). It is unlikely that
degradation of the capsule would profoundly improve the intracellular host environment in such a short time, and we interpret the
burst size increase as an increase in the number of adsorbed phages
that go on to infect cells productively. With this interpretation, the K1
capsule of cells growing in serum normally blocks efcient genome
entry into the cell for over 90% of adsorbed K1-ind phages, but those
phages remain functional (at least in the short term), and the block
can be reversed by capsule reduction either enzymatically or upon
Fig. 3. Fitnesses of phage K1-ind(1) in serum and broth with and without endo-N
supplementation. The diagonal line, at which tness is equal under the two treatments,
is provided only to facilitate visualizing the difference between the two treatments. A
point above the line indicates higher tness with endo-N supplementation than
without. Standard error bars are shown in both dimensions, but may be obscured by the
data point.

over 7 doublings/h (Fig. 3). Furthermore, the effect was specic to

growth in serum, with no appreciable benet in broth.
Phage tails are thought to affect the early part of the phage life
cycle, host recognition and delivery of the genome into the host. Thus
if the tailspike accounts for the consistently lower serum tness of K1ind phages relative to K1-dep phages, the effect should be manifest in
either adsorption or genome entry rates. The effect should be striking
if adsorption was the only trait affected: at a density of 108 cells/mL, a
tness drop from 15 to 10 dbl/h requires a 15-fold drop in adsorption
rate (see Bull, 2006). Although all three tness parameters (adsorption rate, lysis time and burst size) likely change between cells grown
and infected in broth versus serum, there should be a profound
reduction in adsorption or genome entry rate that is common to K1ind phages that is abolished by addition of free endo-N.
Although adsorption rate is the obvious a priori candidate for the
effects of tailspike, it was not obviously affected by endo-N addition.
The adsorption rate in the absence of endo-N (1 10 9 mL min 1)
is already close to the proposed theoretical upper limit (approximately 10 8, Adams, 1959) and could not improve enough to account
for the observed tness gain. Indeed, adsorption rates, measured as
the ratio of unadsorbed to total phages after dilution into 37 C

Fig. 4. Representative one-step growth curve of K1-ind(1) on CAB1. Exponentially

growing cells in serum were allowed to adsorb phage (multiplicity of 0.1) for 5 min,
and then diluted 1000 into either serum (no endo-N, dashed curve) or serum with
endosialidase (+endo-N after adsorption, solid curve). At 60 min, the estimated burst
size for the endosialidase treatment was 500, versus 30 for no-sialidase. Infective
centers were a high enough proportion of total added phage (36% for the endosialidase
treatment, 47% for the pure serum treatment) that the difference in burst size cannot be
attributed to an artifact of sampling error.

Fig. 5. Relative amount of capsular sialic acid in strain CAB1 grown in LB or bovine
serum. (A) Representative chromatographic prole of sialic acid from cells grown in LB
medium. (B) Representative prole of cells grown in serum; the sample size was onehalf that used in panel A. The insert gives the peak area in millivolts min 1 normalized
for cell number for two independent experiments under each growth condition. Data
for the two conditions are different (p = 0.01) by a one-tailed Student's t-test where a
probability b 0.05 was considered signicant.

J.J. Bull et al. / Virology 398 (2010) 7986

growth in broth. Presumably, the addition of endo-N allows adsorbed

K1-ind phages to complete an infection cycle rather than releasing
them into the medium.
These data suggest that cells grown in the presence of serum
possess more capsular material than the same cells grown in broth.
We therefore determined the sialic acid content of CAB1 cells growing
in each medium. A 2.5 increase in the amount of sialic acid was
observed in serum-grown cells (Fig. 5). Note that the absence of
material eluting later than sialic acid means that any phase variation
involving O-acetylation of the sialic acid is not affected by growth in
LB or in serum.
Discussion
Smith and Huggins (1982) concluded that phage therapy success
in a mouse infection depended fundamentally on the type of phage
used. For an E. coli bacterial host of serotype O18:K1:H7, the best
phages required the bacterial capsular K1 antigen for infection (K1dep phages), whereas phages that did not require K1 (K1-ind phages)
were less effective. Smith and Huggins casually suggested that K1-ind
phages were intrinsically poor at growth under all conditions.
Subsequent work with a single phage of each type refuted that
suggestion, showing that a K1-ind phage grew equally well as a K1dep phage when cells were incubated in broth (Bull et al., 2002). K1dep phage appeared to have superior tness when cells were grown
in serum, but the reproducibility and generality of those data required
conrmation.
The goals here have been to determine if the apparent inferiority of
K1-ind phages in therapeutics is general and if so, its basis. In broth,
tnesses of K1-dep and K1-ind phages on the K1-capsulated strain
CAB1 overlapped, but the genome sequences do not even reveal clues
for why tnesses of closely related phages differ. However, a tness
pattern appears to be general in serum, tnesses of K1-ind phages are
lower than K1-dep phages. Thus, in serum medium, an environment
more characteristic of a mammalian host than rich broth media, the
specialist K1-dep phages grow better than phages that can plaque on
both capsulated and non-capsulated strains.
Three phages were intentionally selected for high tness, and
these represent the high-tness tails of the distributions. However,
amplication of K1-ind(2), which was selected for high tness in
serum by serial passage of a mixture of K1-ind phages, was 16-fold
lower than the least t [K1-dep(1)], and 1000-fold lower than the
ttest K1-dep phage [K1-dep(4)] in serum. This difference may have
profound effects in therapeutic applications.
Several observations support the thesis that endosialidase
activity of the tailspike is both the determinant of the K1-dep
phenotype and is necessary for high tness in serum. (1) Of three
K1-ind phages and four K1-dep phages assayed for tness, sialidase
activity of the virion correlated perfectly with the K1-dep
phenotype and thus with high tness in serum. (2) The genome
sequences of all four K1-dep phages reveal that the tailspike protein
contains a sialidase domain whereas tailspike in the three K1-ind
phages is predicted to contain a different glycosidase. Indeed, the
sequence of the tailspike enzyme domain is the only major
difference between some K1-ind and K1-dep phage genomes. (3)
Addition of free endosialidase to a culture of the capsulated strain
CAB1 infected by a K1-ind phage raised its tness substantially, but
only in serum. Serum-grown cells were shown to contain 2.5 more
K1 capsule than when grown in rich broth. The extra capsular
material could be manifest by longer chains of sialic acid, making a
thicker capsule, or more chains that make the capsule more dense.
We favor the latter possibility because longer chains would likely
inhibit the initial adsorption of K1-ind phages to their O-antigen
receptor, but this was not observed. Conversely, a more dense K1
capsule might be expected to inhibit or at least decrease the rate of
O-antigen hydrolysis by K1-ind phages as the entire virion needs to

83

penetrate the capsule in order to reach the cell surface when

genome penetration of the cytoplasm could be initiated.
Merril et al. (1996) evolved two laboratory phages for long term
retention in mice. Enhanced persistence of was due to a single
amino acid change in the viral capsid protein (Vitiello et al., 2005).
This result raises the prospect of using selection in vivo to improve
phage performance. However, selection for faster serum growth of a
single K1-ind phage was unsuccessful; too many simple mutations are
presumably necessary to change the enzyme specicity of a K1-ind
tailspike to a sialidase. Thus, success in selection of a more useful
therapeutic agent by short-term adaptation of a single phage is not
assured. If the original phage has already been carefully characterized,
it should be possible to engineer the desired derivative. Alternatively,
and perhaps more practical as a general approach, selection from a
pool of phages is likely to allow isolation of a superior therapeutic
phage. We have shown here that phages need not be tested
individually to recover those with high growth rate; competition
within a large pool may be used and conditions can easily be chosen
where recombination can occur between different phages in the pool.
Serial passage under selective conditions will then allow isolation of
the best phage. This is, of course, the approach successfully followed
for decades in Eastern Europe, but in the West may result in
regulatory obstacles.
The ramications of this study for use of phages in treating
bacterial infections are threefold:
(1) Bacterial susceptibility to treatment with a particular phage
depends fundamentally on the specic growth environment. In
the context of this work, good phage performance in a host-like
environment requires a specic tailspike enzyme to penetrate
the bacterial capsule. This result underscores the need to test
phage performance in an appropriate environment.
(2) The poor growth rate on a K1-capsulated strain of a phage
possessing a tailspike that degrades O-antigen is not overcome
by short-term adaptation, either as an individual or by allowing
recombination with other phages with the same receptor type.
However, given the genome similarity between some K1-dep
and K1-ind phages, adaptations that allow recombination
between different phage genomes could prove useful.
(3) A broad host range phage is an oft-cited desirable goal for
therapeutic applications. However, the broad host range
phages studied here, those growing on E. coli possessing an
O18 antigen but blind to the presence of a K1 capsule (at least
in broth cultures), are distinctly inferior to the narrower host
range K1 capsule-dependent phages in the environment where
they are likely to be used.
It has commonly been assumed that the success of phage therapy
to treat systemic infections is tied, at least qualitatively, to a phage's
ability to grow within the mammalian host. Assumptions of this
nature can be traced to early reviews of (and doubts about) phage
therapy, for which it was believed that phages could not cure
infections unless they were replicating in vivo (Eaton and BayneJones, 1934; Krueger and Scribner, 1941; Morton and Engley, 1945).
Smith and Huggins (1982) explicitly considered that treatment
success in their model was a function of phage growth in vivo, and
several mathematical models of phage therapy have assumed a direct
relationship between phage growth rate and infection clearance
(Levin and Bull, 1996, 2004; Payne and Jansen, 2001, 2003).
Yet there are several alternatives to phage tness as the main
determinant of treatment success. First, some experimental evidence
supports the view that, when applied in sufcient doses, nonreplicating phage have superior treatment properties over phages
that grow by reducing the release of endotoxins (Hagens et al., 2004;
Matsuda et al., 2005; Westwater et al., 2003). That is, high phage
growth rate can harm the host by releasing toxins too quickly. Second,
and of greater relevance to the present study, is the demonstration

84

J.J. Bull et al. / Virology 398 (2010) 7986

that treatment of E. coli O18:K1:H7-infected mice with endosialidase

alone can cure an infection (Mushtaq et al., 2004). Thus, the in vivo
treatment superiority of K1-dep phages may be partly or largely due
to free enzyme released at lysis rather than growth rate per se. Phage
assembly is typically imperfect in that not all components are
exhausted, so that potentially large numbers of structural components are released as free proteins at lysis. The importance of phage
growth rate versus endosialidase to treatment success could be tested
by either (i) the comparison of treatment success of a K1-ind and a
K1-dep phage with the same growth rate, or (ii) the comparison of
treatment success of two phages differing in growth rate but with the
same type of tail ber. For the collection of phages tested here, only
(ii) is possible; tnesses in serum of K1-dep(4) and K1-dep(1) differ
by over 5 doublings/h (a 50-fold difference) yet their genomes, and
presumably then their mode of infection and development in host
cells, are highly similar.
The question remains of how best to identify, a priori, phages
useful for therapy. If serum proves to be a suitable alternative to an in
vivo environment for systemic infections, and if phage growth rate in
vitro reects in vivo success, then the task is simple. For non-systemic
infections, phage growth in, for example, laboratory-generated
biolms may prove useful. At least for human bacterial infections,
the success of phage therapy may depend on developing appropriate
in vitro environments to select for those phages most likely to function
well in the environment where the infection is focused.

to 10 mL media (125 mL ask, 37 C, 200 rpm in a water bath), grown

for 60 min (LB) or 90 min (serum) to approximately 108 cells/mL.
Serum as medium yields a more variable density at 90 min than LB at
60 min, and cell density at 90 min in serum was often as low as 5 107,
but this variation did not greatly affect the outcome of tness assays.
Fitness assays measure phage growth rate under ideal conditions
of unlimited hosts. Phage were added and grown for 30 min or 60 min
(with phage density remaining well below cell density), at which time
a small aliquot was transferred directly to the next culture, already
grown for the requisite time. The transfer volume included phage and
live (infected and uninfected) cells to preserve the age distribution of
infections. A sample from the completed culture was treated
immediately with chloroform and stored. The recipient ask was
grown for the same length as the rst, and the process repeated as
needed. Fitness was estimated from Pt = P0 2Dt, where t is measured
in hours and D represents doublings/h. P0 was estimated from the
sample of the rst culture, Pt was typically estimated at least an hour
later.
Essentially the same protocol was used for serial transfer as for
tness assays, except that large populations (N105 phages) were
maintained with serial transfer to enhance mutation input and to
avoid stochastic loss of rare types. Phage densities were sometimes
allowed to exceed cell densities near the end of a culture. Serial
transfer was used to (i) adapt phages to specic growth conditions,
and (ii) select the fastest growing phage from a mixture of many
phages.

Methods
Adsorption assays and one-step growth curves
Media
LB (10 g NaCl, 10 g Bacto tryptone, 5 g Bacto yeast extract per liter)
and 100% bovine serum (Bioreclamation Inc., Nicksville, NY) were
used for bacterial growth. The serum was subjected to 0.1 ltration
and stored in bulk at 80 C; aliquots were heat treated (5558 C for
1030 min) within 3 h of use.
Strains
Bacteria CAB1 (E. coli O18:K1:H7) and CAB281 (a K1-chimera of
CAB1 and E. coli K12) were used (Bull et al., 2002). Most phages that
infect CAB281 also infect CAB1 but do not require the K1 capsule for
infection. Phages K1E and K1-5, referred to here as K1-dep(2) and K1dep(3), respectively, were from the collection of IJM; phages LW and
LH were described in Bull et al. (2002), they are referred to here as
K1-ind(1) and K1-dep(1), although they have been formally named
K1ind1 and K1H, both respectively.
Most phage strains used or screened here were initially isolated by
their ability to plate on CAB1 (sometimes on CAB281). Postsedimentation sewage water was obtained either from Walnut
Creek plant, Austin, TX or from the municipal plant in Moscow, ID,
USA. Sewage water, with or without chloroform treatment, was
brought to 1 M NaCl and 10% PEG, and allowed to precipitate
overnight at 4 C. After centrifugation at 3000 rpm for 15 min, the
precipitate was resuspended in LB containing 20% glycerol and stored
at 80 C. Resuspensions were plated directly (without amplication) at 37 C on CAB1 to obtain isolated phages, or were added to
liquid cultures of CAB1 or CAB281 to initiate serial transfer. The only
phages used from Idaho were 21 isolates included in the 54 K1-ind
isolates tested for growth rate in serum on CAB1; the phage with
highest growth rate in this sample, K1-ind(2), was from Austin.
Serial transfer and tness assays
Serial transfer and tness assays followed protocols used in
previous studies (e.g., Bull et al., 2004; Heineman and Bull, 2007;
Heineman et al., 2005). Briey, cells from a frozen aliquot were added

For most assays, cells were grown in serum for 90 min as for tness
assays, and phage added. Phage and cells were plated on LB agar. Cell
density (C) was estimated by plating 12 min before phage addition.
Assays to estimate adsorption rates with and without exogenous
endo-N were performed in parallel. Cells were grown in 10 mL serum
for 75 min, then divided equally into two asks, one of which received
crude endo-N (equivalent to 1010 K1-dep(4) pfu/mL). At 90 min, an
aliquot of the culture was plated to determine cell concentration, and
phage was added to the remainder. Five minutes after phage addition,
each culture was diluted 1000 into pre-warmed serum (with or
without endo-N, according to the treatment) to halt further infection.
Within ten minutes, an aliquot was plated to determine the total
phage titer (free and attached phages, Pt), and the supernatant from
the same aliquot (centrifuged at 10,000 rpm, 3060 s) was plated to
estimate the free, unadsorbed phage (Pu). The adsorption rate (k) was
estimated from Pu = Pt ekCt, where t is the duration of adsorption
(minutes), and C the cell density from the endo-N-free culture.
One-step growth curves were paired between serum-only and
serum + endo-N treatments. Cells grown in serum in a common ask
for 90 min were infected with phage (multiplicity of 0.10.2) for
5 min, then diluted 1000 or more into serum, with or without endoN, to reduce further infection. A few minutes later (before any phage
release), an aliquot from each dilution was plated to determine the
total phage titer (free and attached phages); the supernatant from a
centrifuged sample was also plated to estimate unadsorbed phage.
Together, these two measures provided an estimate of the density of
infective centers. At subsequent times, both before and after the burst,
aliquots were plated to determine the titers of combined free phage
and infected cells. Burst size is given per infective center.
Detection and overproduction of endo-N
The endo-sialidase activity of endo-N (endo-N-acylneuraminidase) was determined as described in Petter and Vimr (1993). Briey,
10 g colominic (polysialic) acid (Sigma) was incubated for 2 h at
37 C with either phage or recombinant endo-N in PBS. The entire
reaction contents (6 L) were spotted on silica gel thin layer plates

J.J. Bull et al. / Virology 398 (2010) 7986

and developed in solvent prior to visualization of degradation

products by staining with orcinol reagent.
Recombinant endo-N was overproduced by IPTG-induction of
plasmid pEndo-N, which harbors the cloned PK1E endo-sialidase
gene, as previously described (Steenbergen and Vimr, 2008). Under
these conditions, approximately 2% of the soluble protein is endo-N.
Enzyme was released from cells by a combination of freeze-thaw and
sonication. The supernatant was used directly at a nal concentration
equivalence of 1010 K1-dep(4) pfu/mL.
Quantication of capsular sialic acid
Overnight cultures of CAB1 started from single colonies were
diluted 1:30 into fresh LB at 37 C and grown to an A600 of about 0.6.
Samples of these cultures were then diluted 1:30 into fresh LB or an
equal volume of heat-inactivated bovine serum and allowed to grow
with aeration for 90 or 120 min in LB and serum, respectively. Cells
were then plated on LB medium to determine their concentration in
colony forming units/mL.
The relative amount of sialic acid was determined by reverse-phase
chromatography of residues derivatized with 1,2-diamino-4,5-methylenedioxybenzene (DMB) essentially as described in Steenbergen et al.
(2006). Briey, cells from 3-mL portions of LB or serum were collected
by centrifugation and washed twice with equal volumes of cold PBS
before a nal wash in water. Pellets from the nal wash were
resuspended in 0.1 mL of water, and an equal volume of 4 M acetic acid
was added prior to hydrolysis for 3 h at 80 C to release total free sialic
acid. Cell debris was removed by centrifugation and the supernatants
concentrated 20-fold by vacuum centrifugation. Samples were
derivatized with DMB and the modied sialic acid were detected by
uorescence after reverse-phase chromatography. Peak areas (millivolts min 1) were calculated and normalized for cell number.
Sequencing and bioinformatics
Genomic DNA of phage isolates was subjected to 454 pyrosequencing and contig assembly by the University of South Carolina
genomics core facility. No attempt was made to establish the physical
ends of genomes and the left genome end was arbitrarily set to the
rst base of the initiation codon for the putative terminase. Orfs and
signicant BlastP similarities are shown in Table S1; the ve genome
sequences have been deposited in GenBank with Accession Numbers
GU196277GU196281.
Acknowledgments
Supported by NIH grants GM57756 (JJB), AI042015 (ERV), and GM
32095 (I.J.M.). Signicant funding was also provided by the Miecher
Regents Professorship to J.J.B. We thank R. Springman for depositing
sequences into GenBank, and S. Abedon, A.D. Ellington and B.R. Levin
for their encouragement.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.virol.2009.11.040.
References
Adams, M.H., 1959. Bacteriophages. Interscience Publishers, New York.
Barbirz, S., Muller, J.J., Uetrecht, C., Clark, A.J., Heinemann, U., Seckler, R., 2008. Crystal
structure of Escherichia coli phage HK620 tailspike: podoviral tailspike endoglycosidase modules are evolutionarily related. Mol. Microbiol. 69, 303316.
Brussow, H., 2005. Phage therapy: the Escherichia coli experience. Microbiology 151,
21332140.
Bull, J.J., 2006. Optimality models of phage life history and parallels in disease evolution.
J. Theor. Biol. 241, 928938.

85

Bull, J.J., Molineux, I.J., 2008. Predicting evolution from genomics: experimental
evolution of bacteriophage T7. Heredity 100, 453463.
Bull, J.J., Regoes, R.R., 2006. Pharmacodynamics of non-replicating viruses, bacteriocins
and lysins. Proc. Biol. Sci. 273, 27032712.
Bull, J.J., Levin, B.R., Derouin, T., Walker, N., Bloch, C.A., 2002. Dynamics of success and
failure in phage and antibiotic therapy in experimental infections. BMC Microbiol.
2, 35.
Bull, J.J., Badgett, M.R., Springman, R., Molineux, I.J., 2004. Genome properties and the
limits of adaptation in bacteriophages. Evolution 58, 692701.
Carlton, R.M., Noordman, W.H., Biswas, B., De Meester, E.D., Loessner, M.J., 2005.
Bacteriophage P100 for control of Listeria monocytogenes in foods: genome
sequence, bioinformatic analyses, oral toxicity study, and application. Regul.
Toxicol. Pharmacol. 43, 301312.
Coates, A.R., Hu, Y., 2007. Novel approaches to developing new antibiotics for bacterial
infections. Br. J. Pharmacol. 152, 11471154.
De Lappe, N., Doran, G., O'Connor, J., O'Hare, C., Cormican, M., 2009. Characterization of
bacteriophages used in the Salmonella enterica serovar Enteritidis phage-typing
scheme. J. Med. Microbiol. 58, 8693.
Denou, E., Bruttin, A., Barretto, C., Ngom-Bru, C., Brssow, H., Zuber, S., 2009. T4 phages
against Escherichia coli diarrhea: potential and problems. Virology 388, 2130.
Eaton, M.D., Bayne-Jones, S., 1934. Bacteriophage therapy. Review of the principles and
results of the use of bacteriophage in the treatment of infections. J Am. Med. Assoc.
103, 17691776.
Ebert, D., 1998. Experimental evolution of parasites. Science 282, 14321435.
Fong, S.S., Nanchen, A., Palsson, B.O., Sauer, U., 2006. Latent pathway activation and
increased pathway capacity enable Escherichia coli adaptation to loss of key
metabolic enzymes. J. Biol. Chem. 281, 80248033.
Gross, R.J., Cheasty, T., Rowe, B., 1977. Isolation of bacteriophages specic for the K1
polysaccharide antigen of Escherichia coli. J. Clin. Microbiol. 6, 548550.
Hagens, S., Blasi, U., 2003. Genetically modied lamentous phage as bactericidal
agents: a pilot study. Lett. Appl. Microbiol. 37, 318323.
Hagens, S., Habel, A., Von Ahsen, U., Von Gabain, A., Blasi, U., 2004. Therapy of
experimental pseudomonas infections with a nonreplicating genetically modied
phage. Antimicrob. Agents Chemother. 48, 38173822.
Hanlon, G.W., 2007. Bacteriophages: an appraisal of their role in the treatment of
bacterial infections. Int. J. Antimicrob. Agents 30, 118128.
Heineman, R.H., Bull, J.J., 2007. Testing optimality with experimental evolution: lysis
time in a bacteriophage. Evolution 61, 16951709.
Heineman, R.H., Molineux, I.J., Bull, J.J., 2005. Evolutionary robustness of an optimal
phenotype: re-evolution of lysis in a bacteriophage deleted for its lysin gene. J. Mol.
Evol. 61, 181191.
Kataoka, Y., Miyake, K., Iijima, S., 2006. Coliphage derived sialidase preferentially
recognizes nonreducing end of polysialic acid. J. Biosci. Bioeng. 101, 198201.
Krueger, A.P., Scribner, E.J., 1941. Bacteriophage therapy: II The bacteriophage, its
nature and therapeutic use. J. Am. Med. Assoc. 116, 21602167.
Leggate, D.R., Bryant, J.M., Redpath, M.B., Head, D., Taylor, P.W., Luzio, J.P., 2002.
Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to
dene the site of proteolytic processing and requirements for catalysis. Mol.
Microbio. 44, 749760.
Leiman, P.G., Molineux, I.J., 2008. Evolution of a new enzyme activity from the same
motif fold. Mol. Microbiol. 69, 287290.
Leiman, P.G., Battisti, A.J., Bowman, V.D., Stummeyer, K., Muhlenhoff, M., GerardySchahn, R., Scholl, D., Molineux, I.J., 2007. The structures of bacteriophages K1E and
K1-5 explain processive degradation of polysaccharide capsules and evolution of
new host specicities. J. Mol. Biol. 371, 836849.
Leverentz, B., Conway, W.S., Camp, M.J., Janisiewicz, W.J., Abuladze, T., Yang, M., Saftner,
R., Sulakvelidze, A., 2003. Biocontrol of Listeria monocytogenes on fresh-cut produce
by treatment with lytic bacteriophages and a bacteriocin. Appl. Environ. Microbiol.
69, 45194526.
Levin, B.R., Bull, J.J., 1996. Phage therapy revisited: the population biology of a bacterial
infection and its treatment with bacteriophage and antibiotics. Am. Nat. 147,
881898.
Levin, B.R., Bull, J.J., 2004. Population and evolutionary dynamics of phage therapy. Nat.
Rev. Microbiol. 2, 166173.
Lu, T.K., Collins, J.J., 2007. Dispersing biolms with engineered enzymatic bacteriophage. Proc. Natl. Acad. Sci. U.S.A. 104, 1119711202.
Machida, Y., Hattori, K., Miyake, K., Kawase, Y., Kawase, M., Iijima, S., 2000a. Molecular
cloning and characterization of a novel bacteriophage-associated sialidase. J. Biosci.
Bioeng. 90, 6268.
Machida, Y., Miyake, K., Hattori, K., Yamamoto, S., Kawase, M., Iijima, S., 2000b.
Structure and function of a novel coliphage-associated sialidase. FEMS Microbiol.
Lett. 182, 333337.
Matsuda, T., Freeman, T.A., Hilbert, D.W., Duff, M., Fuortes, M., Stapleton, P.P., Daly, J.M.,
2005. Lysis-decient bacteriophage therapy decreases endotoxin and inammatory mediator release and improves survival in a murine peritonitis model. Surgery
137, 639646.
Mattey, M., Spencer, J., 2008. Bacteriophage therapy-cooked goose or phoenix rising?
Curr. Opin. Biotechnol. 19, 608612.
Merril, C.R., Biswas, B., Carlton, R., Jensen, N.C., Creed, G.J., Zullo, S., Adhya, S., 1996.
Long-circulating bacteriophage as antibacterial agents. Proc. Natl. Acad. Sci. U.S.A.
93, 31883192.
Morton, H.E., Engley, F.B., 1945. Dysentery bacteriophages. J. Am. Med. Assoc. 127,
584592.
Muhlenhoff, M., Stummeyer, K., Grove, M., Sauerborn, M., Gerardy-Schahn, R., 2003.
Proteolytic processing and oligomerization of bacteriophage-derived endosialidases. J. Biol. Chem. 278, 1263412644.

86

J.J. Bull et al. / Virology 398 (2010) 7986

Mushtaq, N., Redpath, M.B., Luzio, J.P., Taylor, P.W., 2004. Prevention and cure of
systemic Escherichia coli K1 infection by modication of the bacterial phenotype.
Antimicrob. Agents Chemother. 48, 15031508.
Payne, R.J., Jansen, V.A., 2001. Understanding bacteriophage therapy as a densitydependent kinetic process. J. Theor. Biol. 208, 3748.
Payne, R.J., Jansen, V.A., 2003. Pharmacokinetic principles of bacteriophage therapy.
Clin. Pharmacokinet. 42, 315325.
Petter, J.G., Vimr, E.R., 1993. Complete nucleotide sequence of the bacteriophage K1F
tail gene encoding endo-N-acylneuraminidase (endo-N) and comparison to an
endo-N homolog in bacteriophage PK1E. J. Bacteriol. 175, 43544363.
Projan, S., 2004. Phage-inspired antibiotics. Nat. Biotechnol. 22, 167168.
Scholl, D., Rogers, S., Adhya, S., Merril, C.R., 2001. Bacteriophage K1-5 encodes two
different tail ber proteins, allowing it to infect and replicate on both K1 and K5
strains of Escherichia coli. J. Virol. 75, 25092515.
Scholl, D., Kieleczawa, J., Kemp, P., Rush, J., Richardson, C.C., Merril, C., Adhya, S.,
Molineux, I.J., 2004. Genomic analysis of bacteriophages SP6 and K1-5, an estranged
subgroup of the T7 supergroup. J. Mol. Biol. 335, 11511171.
Schoolnik, G.K., Summers, W.C., Watson, J.D., 2004. Phage offer a real alternative. Nat.
Biotechnol. 22, 505506 author reply 506-507.
Smith, H.W., Huggins, M.B., 1982. Successful treatment of experimental Escherichia coli
infections in mice using phage: its general superiority over antibiotics. J. Gen.
Microbiol. 128, 307318.
Soothill, J., Hawkins, C., Anggard, E., Harper, D., 2004. Therapeutic use of bacteriophages.
Lancet Infect Dis. 4, 544545.

Steenbergen, S.M., Lee, Y.C., Vann, W.F., Vionnet, J., Wright, L.F., Vimr, E.R., 2006. Separate
pathways for O acetylation of polymeric and monomeric sialic acids and identication
of sialyl O-acetyl esterase in Escherichia coli K1. J. Bacteriol. 188, 61956206.
Steenbergen, S.M., Vimr, E.R., 2008. Biosynthesis of the Escherichia coli K1 group 2
polysialic acid capsule occurs within a protected cytoplasmic compartment. Mol.
Microbiol. 68, 12521267.
Stummeyer, K., Schwarzer, D., Claus, H., Vogel, U., Gerardy-Schahn, R., Muhlenhoff, M.,
2006. Evolution of bacteriophages infecting encapsulated bacteria: lessons from
Escherichia coli K1-specic phages. Mol. Microbiol. 60, 11231135.
Sulakvelidze, A., 2005. Phage therapy: an attractive option for dealing with antibioticresistant bacterial infections. Drug Discov. Today 10, 807809.
Tomlinson, S., Taylor, P.W., 1985. Neuraminidase associated with coliphage E that
specically depolymerizes the Escherichia coli K1 capsular polysaccharide. J. Virol.
55, 374378.
Vitiello, C.L., Merril, C.R., Adhya, S., 2005. An amino acid substitution in a capsid protein
enhances phage survival in mouse circulatory system more than a 1000-fold. Virus
Res. 114, 101103.
Weiss, M., Denou, E., Bruttin, A., Serra-Moreno, R., Dillmann, M.L., Brssow, H., 2009. In
vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with
Escherichia coli. Virology 388, 2130.
Westwater, C., Kasman, L.M., Schoeld, D.A., Werner, P.A., Dolan, J.W., Schmidt, M.G.,
Norris, J.S., 2003. Use of genetically engineered phage to deliver antimicrobial
agents to bacteria: an alternative therapy for treatment of bacterial infections.
Antimicrob. Agents Chemother. 47, 13011307.