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Copyright © 2002, Bull et al; licensee BioMed
Central Ltd. This is an Open Access article: verbatim copying and
redistribution of this article are permitted in all media for any purpose,
provided this notice is preserved along with the article's original
URL. BMC
Microbiol. 2002; 2 (1): 35
Abstract
Dynamics of success and failure in phage and
antibiotic therapy in experimental infections
J J. Bull, 1
Bruce R. Levin, 2 Terry DeRouin, 2
Nina Walker, 2 and
Craig A. Bloch 3
1Section of Integrative Biology and
Institute for Cellular and Molecular Biology, University of Texas, Austin,
TX 78712-1023, USA 2Department of Biology, Emory University,
Atlanta, GA 30322, USA 3Department of Pediatrics, University of
Michigan Medical School, Ann Arbor, MI 48104, USA
Received August 26, 2002; Accepted November 26,
2002; Published November 26, 2002.
|
Abstract |
|
Background In 1982 Smith and
Huggins showed that bacteriophages could be at least as effective as
antibiotics in preventing mortality from experimental infections
with a capsulated E. coli (K1) in mice. Phages that required the K1
capsule for infection were more effective than phages that did not
require this capsule, but the efficacies of phages and antibiotics
in preventing mortality both declined with time between infection
and treatment, becoming virtually ineffective within 16 hours.
Results We develop quantitative
microbiological procedures that (1) explore the in vivo processes
responsible for the efficacy of phage and antibiotic treatment
protocols in experimental infections (the Resistance Competition
Assay, or RCA), and (2) survey the therapeutic potential of phages
in vitro (the Phage Replication Assay or PRA). We illustrate the
application and utility of these methods in a repetition of Smith
and Huggins' experiments, using the E. coli K1 mouse thigh infection
model, and applying treatments of phages or streptomycin.
Conclusions 1) The Smith and
Huggins phage and antibiotic therapy results are quantitatively and
qualitatively robust. (2) Our RCA values reflect the microbiological
efficacies of the different phages and of streptomycin in preventing
mortality, and reflect the decline in their efficacy with a delay in
treatment. These results show specifically that bacteria become
refractory to treatment over the term of infection. (3) The
K1-specific and non-specific phages had similar replication rates on
bacteria grown in broth (based on the PRA), but the K1-specific
phage had markedly greater replication rates in mouse
serum. |
|
Background |
|
Mounting concerns about drug-resistant pathogenic bacteria [1-3]
have rekindled interest in alternative treatments of bacterial
infections. Prominent among these alternatives is phage therapy, the
use of bacteriophages to kill or otherwise control the bacterial
populations in infected hosts. The use of bacteriophage for the
treatment of bacterial infections is an old idea [4]
that not only caught the imagination of at least one novelist [5]
it was practiced with sporadic successes worldwide in the 1920s and
1930s. However, following the development of antibiotics in the
1940s, the use of phages to treat and prevent infections disappeared
from so-called Western Medicine, but it did survive in the former
Soviet Union. A rekindled interest in phage therapy over the past
decade has inspired historical reviews, increased our awareness of a
substantial body of phage therapy work from Eastern Europe [6-8],
resulted in the West's "discovery" of the Eliava Institute (a
one-time vast and thriving phage therapy research and production
facility in Tiblisi, Georgia [9]),
and motivated the formation of companies developing phage therapy
(Biophage Inc. of Montreal, Canada; Exponential Biotherapies Inc. of
Port Washington, NY; Intralytix of Baltimore, MD). In recent years
this renewed interest in phage therapy has been displayed in
articles in the popular press, (e.g., Kuchment 2001, Superbug
Killers, News Week, Dec. 17, 2001 50–51) and, of course, reviews and
discussions on the internet, http://www.pubmedcentral.gov/redirect.cgi?&&reftype=other&artid=138797&&http://www.phage.org;
http://www.pubmedcentral.gov/redirect.cgi?&&reftype=other&artid=138797&&http://www.evergreen.edu/user/T4/PhageTherapy/Phagethea.html.
Renewed interest in phage therapy is also evident from recent
empirical studies of phage therapy [10-15]
and theoretical excursions into the population dynamics of phage
therapy [16,17].
This rebirth has also revived attention to earlier experimental work
on phage therapy and prophylaxis, including several impressive
studies using phage to treat and prevent bacterial infections in
mice, calves, piglets and lambs by H. Williams Smith, M.B Huggins
and their colleagues [18-20].
The earliest of the Smith and Huggins studies [18]
is especially instructive in this light. Using experimental, lethal
infections of an E. coli 018:K1:H7 into the mouse thigh, it
showed: (i) phage were at least as effective as antibiotics in
preventing mouse mortality; (ii) not all phages were equally useful,
rather those that required the K1 antigen for infection were
superior to phages not requiring K1; (iii) survival declined with a
delay in treatment, even though the treatment was applied well
before untreated mice had overt symptoms of the infection. Perhaps
most importantly, Smith and Huggins [18]
also provided information about the dynamics of phage treatment –
the changes in bacterial and phage densities over the course of the
infection. Ultimately, the efficacy of antibacterial therapy has to
be measured by the rate at which it eliminates the symptoms of the
infection. Microbiological data on the course of infections with and
without treatment – the population dynamics of the treatment process
– provide a means to understand how treatment operates and allows
one to compare, modify and improve therapeutic protocols.
The present study is offered in the spirit of continuing the
precedent set by Smith and Huggins for analyzing the population
dynamics of phage and antibiotic therapy. We use their infection
model to develop and illustrate the use of two quantitative methods
that facilitate understanding, comparing and developing methods of
antibacterial therapy and protocols for their application. One
method provides a facile measure of efficacy of treatment that is
independent of the clinical outcome of infection and can be applied
on a tissue- and time-specific basis for any form of treatment to
which bacteria can acquire resistance. The other method is a simple
procedure to assay phage growth rate in vitro that can be used to
screen phages to evaluate their potential for
therapy. |
|
Results |
|
Smith and Huggin's results are
repeatable We repeated the experiments of H. William Smith
and M.N. Huggins [18].
Despite the fact that we used different sources of mice, E. coli
018:K1:H7, bacteria and phages, our treatment and mortality rate
results were similar to theirs both qualitatively and quantitatively
(Fig. 1):
(a) All 15 untreated/control mice died within 40 hours of
inoculation with CAB1. All 15 mice survived when treated immediately
with LH (the phage
requiring the bacterial K1 antigen for adsorption) or when treated
immediately with a single dose of 60 μg/gm streptomycin. In
contrast, only 6 of the 15 mice survived when treated immediately
with LW (the phage
that was not specific for the K1-capsule). These results closely
match those of Smith and Huggins [18],
except that they did not report experiments of immediate treatment
with streptomycin or other antibiotic.
(b) Even though mice with untreated infections normally survived
at least 24 hours, delaying treatment for only 8 hours significantly
reduced the rate of survival of mice treated with a single dose of
60 μ/gm and of 100 μ/gm streptomycin. The survival rate of these
streptomycin-treated mice, however, significantly exceeded that of
the controls and that which Smith and Huggins [18]
obtained with a single dose of 25 μ/gm streptomycin. The survival of
mice treated with LH
was slightly reduced with delayed treatment (11 of the 12 mice
treated at 8 hours survived, versus 15 out of 15 with immediate
treatment), a difference that is not statistically significant. In
the Smith and Huggins study, the decline in survival at 8 hours was
significant for the combined samples of 9 isolates of K1-specific
phages, but not for their most efficacious phage. We did not conduct
delayed treatment experiments with LW or with multiple doses
of streptomycin.
The Resistance Competition Assay
(RCA)
Derivation The principle of this
method is that, when a mixture of resistant and sensitive cells is
treated, the relative frequency of bacteria resistant to treatment
will increase at a rate according to efficacy of the treatment. That
is, resistant cells increase relative to sensitive cells by the
amount that treatment kills or inhibits sensitive bacteria. The
derivation of our measure of this efficacy of treatment, RCA,
follows from standard population genetics models of selection
between two haploid genotypes that different in their relative
fitness [21].
(Following convention, we use italicised RCA for the
parameter value or estimate, and normal font RCA for the assay
procedure.) In this perspective, sensitive and resistant bacteria
are the two competing genotypes. The RCA estimates the relative
fitness advantage (or disadvantage) of the resistant bacteria over
sensitive bacteria. In situations where one has the luxury of many
sampling points, an RCA can be calculated from the slope of
the natural log of the ratio of the densities of Resistant/Sensitive
as a function of time [22].
In situations where only two points are feasible, the initial
frequency of resistant bacteria in the inoculum
(p0) and the frequency of resistant bacteria at
time t (pt) an RCA can be calculated
from
RCA = ln [(pt
(1-p0))/(p0(1-pt))]/t
(1)
(for the derivation see Crow and Kimura [21]).
The value of RCA is the selection coefficient of resistant
cells (in the vernacular of population genetics). If RCA >
0 then the resistant cells have an advantage, if RCA < 0,
the resistant cells are at a selective disadvantage. RCA
operates on an exponential scale:
pt/(1-pt) = etRCA
(p0/(1-p0).
(2)
If the differential success of sensitive and resistant cells is
constant over time, then the ratio of resistant to sensitive cells
increases by a factor of eRCA every hour (unit of
t). Assuming that cells are not growing and that treatment kills
sensitive cells, an RCA of 0 means that sensitive cells are
not killed, an RCA of 1 means that 63% of sensitive cells are
killed per hour (95% in 3 hours), and an RCA of 2 means that
86.5% of sensitive cells are killed per hour (99.8% in 3 hours). If
the process is not constant, then the RCA is an average over
the interval of estimation and should not be projected to longer
intervals; however, estimations of RCA at different times
would reveal how treatment efficacy is changing with time.
As an alternative to using the initial frequency of resistant
cells as the estimate of p0, one can substitute
the final frequency (qt) of resistant
bacteria in untreated controls (as was done here). This substitution
controls for intrinsic differences in fitness of the two bacteria,
for differences in the physiological states of the bacteria at the
time of inoculation that could affect their growth rates independent
of treatment, and for possible differences in the host response with
and without treatment. The resulting RCA is thus the
advantage of resistant cells over sensitive cells relative to their
advantage in the absent of treatment. For example, in the absence of
antibiotic treatment, resistant bacteria may have a disadvantage in
competition with sensitives [23],
and use of qt in place of p0
will correct for this effect. It is important, however, that
the controls from which qt is obtained be
inoculated with the same mix of bacteria and be sampled at the same
times as the treated mice.
Bacteria become refractory to phage and
antibiotics within 8 hours As noted in Figure 1
and in more detail in [18-20],
the survival rate from treatment with phage and antibiotics declines
dramatically with the term of infection. A declining efficacy of
delayed treatment is also evident by comparing RCA values for
LH or streptomycin
(Table 1).
The RCA for a single dose of streptomycin was 2.1 after
immediate treatment but declined to 0.5 when treatment was delayed 8
hours. The corresponding values for the K1-specific phage were 1.7
and 0.3. The RCA for LH at 8 hours is statistically indistinguishable from that
for immediate treatment with LW (0.2), even though the mouse survival under these two
conditions differs significantly (Fig. 1;
P < 0.02). It is thus evident that by eight hours the bacteria
had declined in their susceptibility to phage, and antibiotic
efficacy declined as well. It is noteworthy that there were no
apparent clinical symptoms of these infections at 8 hours (or even
at 12 hours).
The bacteria inoculated into the thigh not only became refractory
to antibiotic and phage treatment within a few hours after
inoculation, they persisted and remained refractory to phage
treatment for a number of days if the mouse survived. In samples
taken 7 days after treatment, the density of the LH phage in the thigh was
109 phage particles per gram, and while phage resistant
cells were present, 80% (0.81 ± 0.01) of the bacteria isolated from
leg tissue remained sensitive to this phage.
The Phage Replication Assay: phages LW and LH have similar growth
rates in broth but not serum Mortality rates of mice in
Smith and Huggins [18]
and here, as well as the RCA values, indicate that phages
specific for the K1-capsule are more effective in controlling the
infections than phage that are not specific for the capsule. Could
this have been anticipated from the in vitro capacity of the phage
to replicate on and kill E. coli 018:K1:H7? The Phage
Replication Assay (PRA) attempts to provide a measure of in vitro
efficacy that can be extended to in vivo performance. The rate of
doubling of a population of lytic phage on a population of bacteria
measures the ultimate efficacy of that phage in killing those
bacteria. This overall replication rate depends on adsorption rate,
latent period, and burst size on that bacterial host, as well as
temperature and other physical properties of the culture conditions
(see Additional
file 1). The rate of replication also depends on the ratio of
phage to bacteria (the multiplicity of infection) and most
critically on the density and physiological state of the bacterial
population [24,25]).
Despite the many factors that influence phage replication rates (and
hence influence the PRA value), these factors are easily
controlled, and furthermore, the comparison of PRAs between
different phages is straightforward if the PRAs are obtained
from common bacterial cultures (as here, see Methods).
Our PRA values are surprising in some respects yet are
consistent with the mouse mortalities and microbiological data. When
the PRA of these phage were estimated on cells grown in Luria
Broth, there was no apparent difference between the two phages (Fig.
2);
the PRAs were strongly affected by cell densities, but both
phages showed similar patterns, and there is no basis from these in
vitro data for suggesting any difference in their efficacy at
replication on and killing CAB1. Note that the observed PRAs
declined with increasing bacterial density, the opposite of the
effect seen in the simulations of Additional
file 1. There are in fact two opposing effects of cell density
on the PRA. One is that higher cell densities reduce phage
generation time by decreasing the time for a phage to encounter a
host. This increases the PRA. The other is that high cell
densities exhaust nutrients in the media and thereby slow bacterial
growth, in turn reducing phage metabolism and thus reducing the
PRA. Only the former effect was considered in the model of Additional
file 1.
When the PRA was estimated on cells grown in mouse serum,
the K1-specific phage LH had a much higher rate of replication than LW (Fig. 2).
Despite variance in the estimates, there was a pronounced
superiority of the K1-specific phage LH over the non-specific
phage LW. Although
this serum assay did not fully mimic in vivo growth conditions, it
certainly supports the hypothesis that the greater treatment
efficacy of LH is
specific to phage replication on bacteria in mice and is not from a
general superiority of LH. |
|
Discussion |
|
Our goal here was to develop procedures to explore the dynamical
underpinnings of success and failure in treating bacterial
infections. To illustrate the application of these procedures, we
used the E. coli K1 mouse thigh infection model of Smith and
Huggins [18],
and like them, applied treatments of phages and antibiotics. As part
of this effort, we repeated their experiments to ascertain if their
results were robust. Across a variety of treatments and conditions,
mouse mortality rates were remarkably similar between our study and
theirs, even though we used a different strain of mice, phages from
different sources, and an independently isolated strain of E.
coli 018:K1:H7. Thus, in the absence of treatment, inoculations
of 108 bacteria were fatal in over 95% of mice, but
immediate treatment with phage that were specific for the K1 capsule
or with streptomycin essentially eliminated mortality from the
infection. Immediate treatment with phages that were not specific
for the capsule also reduced mortality, but mortality rates for
treatment with these phage were greater than with the K1-specific
phage. Both studies also observed that mortality increased if
treatment was delayed by 8 hours, under most forms of treatment.
Factors affecting treatment
success Two observations from these combined studies seem
especially interesting. (i) Phages vary in treatment success. (ii)
Delaying treatment by 8 hours substantially increases the rate of
mortality relative to that of immediate treatment (for many of the
treatments applied here), despite the fact infected mice survive at
least 16 hours longer. In attempting to better understand these
observations, we developed two assays. One assay, the RCA
(Resistance Competition Assay) measures the
efficacy of a treatment in killing or inhibiting the growth of
bacteria based on changes in the frequency of treatment-resistant
bacteria after treatment is applied. The other assay, the PRA
(Phage Replication Assay), measures the ability
of a phage to replicate on populations of a bacterium under
controlled, in vitro conditions.
The RCA and PRA values reflect what is observed
with other (microbiological) measures of the efficacy of treatment.
Consider first the effect of delayed treatment, which reduced mouse
survival in most treatments. Increased mortality with delayed
treatment could have various causes: (i) By the time of treatment,
the numbers of bacteria are already sufficiently high that mortality
is caused by toxins and tissue damage ensuing from them, rather than
from further growth of the bacteria. Under these conditions,
reducing the number of bacteria by treatment would have little
effect on the clinical outcome of the infection. (ii) By the time of
treatment, the numbers of bacteria are so high that the mouse's
defenses cannot prevent further growth of the bacterial population,
even with treatment, and bacterial numbers simply increase to the
point of mortality. This model is similar to that in (i), except
that a treatment which controlled bacterial growth would prevent
mortality. (iii) As a consequence of the host's response to the
proliferating population of bacteria, the site of the infection
becomes less accessible to the antibiotic or phages, and the
proliferation of the bacterial population can no longer be
controlled by these antibacterial treatments. (iv) The bacteria
become physiologically refractory to the treatment.
The RCA specifically supports the latter two models. If by eight
hours the bacteria were as accessible and responsive to the phage or
antibiotic as they were initially, the RCA values would
remain high. We instead observed a drop in RCA values. The
data do not rule out additional processes consistent with models (i)
or (ii), but the data clearly reveal a reduced bacterial
susceptibility or reduced access of treatment to the bacteria when
treatment is delayed.
More than a half a century ago, H. Eagle and colleagues observed
a dramatic decline in the efficacy of antibiotics with the term of
the infection in a mouse thigh model. In their now classical
investigations of the within-host dynamics of antibiotic treatment,
they followed the course of penicillin treatment of Treponema
pallidum and Group A and Group B Streptococcus [26-28].
Eagle [28]
postulated that the most likely reason for the declining efficacy of
treatment with the term of the infection was due to (i) a decline in
the rate of metabolism and replication of bacteria during the
infection, and (ii) that slowly growing or non-growing bacteria are
more refractory to penicillin than those that replicating at higher
rates. More recent work by E. Tuomanen, A. Tomaz and their
colleagues supported this interpretation, a phenomenon they called
phenotypic tolerance [29-31].
They also provided evidence that nutrient limitation was the cause
of the decline in the rate of bacterial growth [32].
Not only do bacteria become increasingly refractory to the majority
of antibiotics as their rate of growth declines, but they also
adsorb to and replicate bacteriophage less efficiently [24,33],
as is evident from the PRA values in Fig. 2.
Thus the decline in the efficacy of phage and antibiotic therapy
with delayed treatment in our experiments is plausibly attributed to
a decline in the rate of replication of E. coli K1 once
inside the mouse.
The other intriguing observation from Smith and Huggins that we
corroborated here is the consistent superiority of phages requiring
the K1 antigen for infection, when measured as mouse survival. Our
RCA values showed a significant difference between the phages
in vivo, suggesting that the K1-specific phages replicate at a
higher rate than the non-K1-specific phages inside the mouse. Yet
although Smith and Huggins casually observed that their
non-K1-specific phages were inferior to K1-specific phages at lysing
cultures of bacteria grown in artificial media, suggesting intrinsic
differences between the phages more generally, our PRA
estimates indicated that there was no intrinsic difference
between our two phages in artificial media. Instead PRA
differences were consistent with mouse survival and RCA
data only when the PRA was measured on cells grown in
mouse serum. Thus the PRA enables one to begin unravelling
the environmental bases of differences in phage growth in vivo.
In this investigation, we used the mouse thigh infection model
because of the many precedents for its use and because of its
established repeatability [34-37].
The fact that bacteria undergo such a profound change in
susceptibility or accessibility to treatment in only 8 hours raises
the question of whether the mouse thigh infection model is
representative of natural infections. This question is not
necessarily answerable at present, but the results do highlight the
fact that development of specific protocols and phages for treatment
in any one experimental model may be inadequate for treatment of the
same bacterium under field conditions.
The Resistance Competition Assay The
preceding discussion indicates that the resistance competition assay
is consistent with other measures of phage performance in vivo and
provides specific insights not easily obtained in other ways. The
RCA has other virtues that make it useful for studying phage therapy
and other forms of treatment. (1) It is versatile. In addition to
being used to study the population dynamics of phage and antibiotic
treatment in general, the RCA could be employed to design and
evaluate the efficacy of different antibiotic treatment protocols.
It can be applied to virtually any experimental model, such as
enteric infections [38,39]
or urinary tract infections [40].
(2) The RCA provides a more direct measure of the in vivo action of
antibiotics (or other treatments) than estimates of the
concentrations of these compounds in serum or in solid tissue.
Moreover, the RCA protocol controls for the contribution of the host
defenses as well as variety other factors that could influence the
density of bacteria in a particular tissue. Only the action of the
treatment per se can account for the difference in the frequency of
resistant bacteria between treated and untreated treated hosts. (3)
The RCA is more humane and offers greater statistical power (per
animal) than "outcome" measures of the efficacy of treatment based
on survival or other clinical indications. The fact that the RCA
yields a continuous statistic, rather than a binary one, enables the
use of standard statistical analyses in which meaningful comparisons
can be made with as few as two replicates per group. (4) The RCA can
be applied to populations of bacteria infecting specific tissues at
specific times, even allowing multiple measures per animal. However,
individual tissues subjected to the RCA must not have high levels of
bacterial migration from other tissues over the course of
treatment.
The RCA is a sensitive and specific measure of treatment
efficacy, and as such, will not correlate perfectly with other
measures of treatment. To wit, our RCAs were similar between
delayed LH treatment
(0.3) and immediate LW
treatment (0.2), even though mouse survival rates were significantly
different between them (11 of 12 mice versus 6 of 15). These
discrepancies highlight the complexity of the infection process and
the fact that different measures of infection dynamics capture
different properties. Because our RCAs were based on samples
from the infected thigh, the values apply specifically to that thigh
and do not necessarily reflect the efficacy of treatment in other
tissues that may influence host survival.
The Phage replication assay This
method has a potential utility beyond that demonstrated here. If
phage therapy is to be developed for particular infections, it would
be useful to have an in vitro procedure to screen phages for their
potential efficacy in vivo. The diversity of phage is enormous. For
example among 40 phage isolated from different samples or different
plaques on lawns of E. coli K12 and E. coli B, at
least 32 distinct phage were found [41]
(as measured by host range and/or restriction pattern). A far
greater number of phage could certainly be isolated with a broader
array of lawn bacteria and by sampling different sources. The
implication is that it should be possible to isolate a substantial
number of lytic phage capable of killing most strains of enteric and
other bacteria, hence offering a compelling reason to pursue phage
therapy as a solution to antibiotic resistance. Screening many
phages for their therapeutic potential in experimental animals would
be a time- and animal-consuming task. The PRA employed here could
facilitate that screening. Our results suggest that phage
performance in serum could be a sufficient indicator of in vivo
performance, but there is no reason that this assay could not be
performed in vitro with other modifications, such as solid tissues.
Moreover, as suggested in a recent study [12],
in vivo culture may selectively improve the capacity of a phage to
replicate on and kill bacteria in a mammalian host.
Prospects for phage therapy This is a
methodological study to develop and experimentally evaluate
procedures to measure the efficacy of antibacterial treatment and to
screen bacteriophage for their therapeutic potential. Our purpose in
performing these experiments and publishing these results is not to
advocate the use of phages for the treatment of systemic infections.
Nevertheless, because of the current novelty of and aspirations for
phage therapy as an alternative to antibiotics, it seems appropriate
to acknowledge that our results and those of several other studies
offer promise that phage therapy is highly repeatable, can be
successful, and is thus worthy of further research for clinical
practice. Moreover the use of phage for therapy and prophylaxis
needn't be restricted to humans, as phage could obviously be used
for these purposes in domestic animals.
There are, of course, a number of problems associated with the
use of phage as an alternative to antibiotics. To us the most
serious biological problem is a restricted host range. Not only
would one have to know the species of bacteria responsible for an
infection, it would be necessary to know which phages can infect
that strain of bacteria. These requirements are certainly
inconsistent with current empiric therapy that uses broad spectrum
antibiotics, which dominates how antibiotics are employed in the
community as well as in hospitals. Commonly, it is not clear whether
a bacterial infection is responsible for the symptoms being treated
with antibiotics, much less the species, strain, and resistance
profile of the bacteria responsible. On the other hand, there are
situations, like epidemics, where this knowledge would be available.
And as procedures to identify the bacteria responsible for
symptomatic infections get better and more rapid, it soon may be
quite easy to get this information from individual patients. As a
consequence of the ever increasing frequency of antibiotic resistant
bacteria, the range of antibiotics to which individual bacteria are
resistant, and the limited number of targets to which current (and
soon to be anticipated) antibiotics are directed, there is a
pressing need to develop alternative methods of treating and
preventing bacterial infections. Phages certainly offer some of the
most readily-available and promising
alternatives. |
|
Conclusions |
|
(1) The results of the Smith and Huggins 1982 study of phage and
antibiotic therapy [18]
are repeatable and robust quantitatively as well as qualitatively.
Using their almost invariably (more than 95%) lethal E. coli
K1 mouse thigh infection protocol, but with different strains of
mice and independently isolated phages and bacteria, we obtained the
same frequencies of treatment survival as they did. As they also
observed, phages that required the K1 capsule for infection provided
greater mouse protection than phages that did not require K1. When
treatment was administered within minutes of the infection, both the
K1 specific phage and single doses of streptomycin completely
prevented infection-induced mortality. If, however, treatment was
delayed by eight hours, the rate of recovery with a single dose of
streptomycin (the most effective antibiotic in the Smith and Huggins
study) was reduced by approximately 50%.
2) The Resistance Competition Assay (RCA), one of two protocols
developed here, uses the change in the frequency of a minority
population of bacteria resistant to the treating agent as a measure
of the efficacy of treatment in vivo. Our RCA estimates from
phage and streptomycin treatment of E. coli K1 infections in
laboratory mice are consistent with mortality rates. With immediate
treatment, the RCA values for the streptomycin and
K1-specific phages (which totally prevented mortality) were both
substantially greater than that of the phage not specific for the K1
capsule (for which 60% of the treated mice died). RCA values
for the K1-specific phage and for single doses of streptomycin when
treatment was delayed by eight hours were substantially and
significantly less than those estimated for immediate treatment. The
RCA supports earlier observations that as time between infection and
treatment increases, bacteria become increasingly refractory to
treatment (e.g., because of changes in bacterial physiological
and/or the host environment).
3) The Phage Replication Assay (PRA) provides a simple measure of
the rate of replication (and host cell killing) of lytic phage under
defined conditions. By varying those conditions, it is possible to
identify factors that affect phage growth and hence their efficacy
in killing the target bacteria. Over a wide range of densities of
bacteria growing in artificial medium, the estimated PRAs
were similar between the K1-specific and non-specific phages. Based
on these estimates we would not have anticipated different
efficacies of these phage in treating E. coli K1 infections. On the
other hand, the assay performed in mouse serum yielded substantially
greater efficacy of the K1-specific phage, matching its superior
performance in the mouse.
4) The results of this investigation, like those of Smith and
Huggins and others, support the potential of phage for treating
bacterial infections and the development of experimental infection
models to evaluate and optimize the efficacy of antibiotic as well
as phage treatment protocols. Our results illustrate the value of
exploring the dynamics of the bacterial population during treatment
in the evaluation and design of treatment
protocols |
|
Methods |
|
Culture and sampling medium and
procedures In vitro, liquid cultures of bacteria and phage
were grown and maintained in Luria – Bertani broth (LB),
supplemented with 1 gm/l glucose. Bacterial densities were estimated
from colony counts on Petri dishes (plates) containing 25 mL of LB
with 1.75% agar. Phage densities were estimated on these plates with
3 ml top (0.7%) agar containing LB glucose and 100 ul of an
overnight culture of the bacteria (about 2 × 109 bacteria
per ml). When needed, the bacterial and phage suspensions were
serially diluted in 0.85% saline or LB Glucose before plating. When
rare (less than 10%), phage-resistant bacterial densities were
estimated by plating with and without ~109 phage
particles in soft agar; when common, they were estimated by
streaking individual colonies across high densities of phage on
plates. A similar selective plating procedure was used to estimate
the densities of antibiotic resistant bacteria.
Bacteria The primary strain used in
these experiments was CAB1, an E. coli of the same serotype
as that used by Smith and Huggins [18]
(O18:K1:H7), but isolated from a different source [42].
We also used a TcR, Kps-, E. coli K-12 chimera of this
isolate, designated CAB281, that does not express the K1 antigen [42].
Phage Phages were isolated from an
Atlanta, Georgia sewage treatment plant. An aliquot of liquid sewage
from the inflow to the plant was treated with chloroform to kill
bacteria and human viruses. The chloroform-free supernatant was then
enriched for E. coli 018:K1:H7-specific phage by adding 1 ml
of this suspension to 10 ml LB containing CAB1 (approximately 4 ×
107 bacteria per ml). These mixtures of bacteria and
sewage were grown for a minimum of four hours, mixed with
chloroform, and centrifuged to remove debris. The vast majority of
phages in these crude lysates were able to grow on CAB281 as well as
CAB1, hence were not K1-specific. To enrich for K1-specific phage,
the mixed lysate was incubated with CAB281 and treated with
chloroform 10 minutes later to abort infections. After removing the
chloroform, the cycle was repeated 4 times. The phage were then
plated with lawns of CAB1 and single plaques were patched onto lawns
of CAB281. Of the 50 plaques isolated after this passage, two were
from phage that grew on CAB1 but not on CAB281, hence were presumed
to be specific for the K1-capsule. From these plates, one clone each
of a K1-specific and non-K1 specific phage was chosen, designated
LH and LW, respectively.
The two phages were characterized morphologically and
molecularly. Electron micrographs revealed that both had a B1
morphology [43]
similar to phage λ but with a tail of hexagonal symmetry and a
somewhat shorter head-tail connector than λ. Whole genomes of both
phages migrated in agarose gels at approximately 40 kb; genomes of
LH and LW were sensitive to Xmn
I digestion but showed different digestion patterns.
In vivo experiments All mouse
experiments were conducted at Emory University using protocols
approved by the Emory University Institutional Animal Care
Committee. All of the in vivo experiments were performed with
female, outbred, white (Swiss) mice (Harlin Sprague) of from 6 to 10
weeks of age (22–30 grams). Mice were maintained in cages containing
5 or fewer animals. Using 1 ml tuberculin syringes, suspensions of
bacteria were injected into a mouse thigh. For treatment, phage
lysates, pseudolysates (phage-free bacterial cultures treated with
chloroform), saline or streptomycin sulfate (SIGMA™) solutions were
injected into the opposite thigh or a forelimb muscle. Although
chloroform was absent from injected lysates and pseudolysates, no
other attempts were made to refine these preparations. Except as
noted, the numbers of bacteria and phage injected were each
approximately 108 and the total volume injected was
normally 0.1 ml or less. Following the initial inoculation, the
infected animals were periodically observed (usually at intervals of
less than 12 hours), with more frequent observations (2 hour
intervals) made during the 28–40 hour post infection period when
mortality was anticipated. Infected mice surviving beyond 48 hours
never succumbed to the infection in the next 5 days. In situations
where the mouse's ataxic appearance indicated that death was
imminent, the mouse was euthanized.
Samples of bacteria and phage taken from leg muscle were
maintained for a maximum of 1 hour on ice before being weighed and
suspended in 2 ml of saline and homogenized with a Tissue Tearor™.
The densities of bacteria and phage in these homogenates were
estimated by diluting and plating.
Resistance Competition Assay
(RCA) This assay measures the efficacy of phage or
streptomycin in limiting replication by an infecting population of
bacteria in infected mice. The principle underlying this assay is
that bacteria resistant to a treatment will increase in frequency
over sensitive bacteria only to the extent that the treatment is
effective at killing or reducing the rate of growth of the sensitive
bacteria. The assay converts the advantage of resistance into a
measure of treatment efficacy. For this assay, mice were inoculated
with mixtures of 108 bacteria (CAB1) that were sensitive
to the treatment (phage or antibiotics) along with low frequencies
(10-2–10-3) of bacteria resistant to the
treatment. The mice were then treated with phage or streptomycin
either immediately (0 hours) or after a delay of 8 hours, or were
inoculated with sterile 0.85% saline (controls). At 3, 4, or 4.5
hours after treatment, the mice were sacrificed and the relative
frequencies resistant bacteria in the mixtures were estimated from
the homogenized leg tissue. We applied this assay in two ways: 1) By
using different mice for the immediate and delayed treatments or, 2)
by inoculating the same mouse in different hind limb thighs, with
the second inoculation delayed 8 hours, and treating the mouse
immediately by inoculation of streptomycin, phage or saline into the
musculature of a forelimb
Phage Replication Assay (PRA) This
procedure estimates in vitro the potential therapeutic efficacy of
phages. It simply measures the rate at which a phage population
increases in density (its rate of replication) when grown on a
strain of bacteria under prescribed, controlled conditions. Phage
replication in vivo is presumed to be an integral part of phage
therapy success, so to the extent that replication in vitro mirrors
replication in vivo, this assay should indicate which phages are the
best choices for therapy. The rate of replication of LH and LW on CAB1 was measured in
LB and in mouse serum. Assays in LB were conducted as follows. The
bacteria were grown with aeration in LB at 37° until reaching a
specified density as measured by light scattering from a side-arm
flask. Glucose and calcium were not added to the LB in these assays.
Aliquots of 1 ml of the growing bacterial cultures were added to
empty tubes along with the phage. The suspensions were grown for 1
hr at which time chloroform was added. Assays in mouse serum were
conducted similarly, except that the densities at the time the phage
were added were determined by plating and thus could not be
standardized between replicates. The bacteria were allowed to
replicate in serum for at least 2 hours before addition of phage.
Phage concentrations were determined at the beginning and end points
to calculate a per-capita increase ratio, ρ; we then transformed
this value to log2(ρ) to estimate of the number of
doublings per hour (see the Additional
file 1 for the sensitivity of this measure).
Several precautions were taken to avoid biases and reduce the
error in estimating ρ. Phage concentrations were maintained at low
levels throughout the assay so that uninfected cells did not become
limiting (the final phage density was never greater than the cell
density, indicating that a majority of cells remained uninfected
during the assay period). To reduce sources of variance in growth
rate extrinsic to the phages, growth rate assays of both phages were
usually conducted simultaneously with aliquots of the same parent
culture for both phages. |
|
Authors'
contributions |
|
JB and BL derived the methods used in this paper and conducted
the experiments reported in Fig. 1;
BL created the Additional
file 1. JB conducted the PRA measures in Fig. 2.
BL, NW, and TD carried out the RCA assays of Table 1.
CB provided the bacterial strains CAB1 and CAB281 and advice. All
authors approved the final manuscript. |
|
Acknowledgements |
|
We thank Rustom Antia, and John Mittler for useful suggestions
and Holly Wichman for comments on the manuscript. We are
particularly grateful to Theresa Abak and her colleagues at the
Emory University Animal Care facility for their excellent help and
support in this endeavor. Finally, we wish to acknowledge the
technical assistance and agar engineering of Candice Gianelloni and
Lisa Kelcourse. This investigation was supported by a grant from the
National Institutes of Health to BRL (GM33782) and grants from the
NSF (DEB9726902) and NIH (GM57756) to JJB. We dedicate this report
to the memory of H. Williams
Smith. |
|
References |
|
- Neu, HC, Duma, RJ, Jones, RN, McGowan,
JE, Jr, TF, O'Brien, Sabath, LD, Sanders, CC, Schaffner, W,
Tally, FP, Tenover, FC, et
al. : Antibiotic resistance.
Epidemiology and therapeutics. Diagn Microbiol Infect Dis 1992, 15:53S60S.[PubMed]
- Cohen, ML: Epidemiology of drug resistance: implications for
a post-antimicrobial era. Science 1992, 257:10501055.[PubMed]
- Cohen, ML: Antimicrobial resistance: prognosis for public
health. Trends in
microbiology 1994, 2:422425.[PubMed]
- D'Herrelle, F: The Bacteriophage and Its Belhavior. Baltimore Williams and Wilkins 1926:.
- Lewis, HS: Arrowsmith. New
York Signet Classics – New American Library 1925:.
- Ho, K: Bacteriophage Therapy for Bacterial Infections:
Rekindling a Memory from the Pre-Antibiotics Era. Perspectives in Biology and Medicine
2001, 44:116.[PubMed]
- Sulakvelidze, A, Kekelidze, M,
Gomelauri, T, Deng, Y, Khetsuriani, N, Kobaidze, K, De Zoysa, A,
Efstratiou, A, Morris, JG, Jr, & Imnadze, P: Diphtheria in the Republic of Georgia: use of
molecular typing techniques for characterization of
Corynebacterium diphtheriae strains. J Clin Microbiol 1999, 37:326570.[PubMed][Free
Full Text]
- Sulakvelidze, A, Alavidze, Z, &
Morris, JG, Jr: Bacteriophage
therapy. Antimicrob Agents
Chemother 2001, 45:64959.[PubMed][Free
Full Text]
- Radetsky, : The Good Virus. DISCOVER 1996:.
- Soothill, JS: Treatment of experimental infections of mice with
bacteriophages. J Med
Microbiol 1992, 37:25861.[PubMed]
- Soothill, JS: Bacteriophage prevents destruction of skin grafts
by Pseudomonas aeruginosa. Burns 1994, 20:20911.[PubMed]
- Merril, CR, Biswas, B, Carlton, R,
Jensen, NC, Creed, GJ, Zullo, S, & Adhya, S: Long-circulating bacteriophage as antibacterial
agents. Proc Natl Acad Sci
USA 1996, 93:31883192. [ Free Full text
in PMC]
- Barrow, PA & Soothill, JS:
Bacteriophage therapy and prophylaxis:
rediscovery and renewed assessment of potential. Trends Microbiol 1997, 5:26871.[PubMed][Full
Text]
- Barrow, P, Lovell, M, & Berchieri,
A, Jr: Use of lytic
bacteriophage for control of experimental Escherichia coli
septicemia and meningitis in chickens and calves. Clin Diagn Lab Immunol 1998, 5:2948.[PubMed][Free
Full Text]
- Biswas, B, Adhya, S, Washart, P, Paul,
B, Trostel, AN, Powell, B, Carlton, R, & Merril, CR:
Bacteriophage therapy rescues mice
bacteremic from a clinical isolate of vancomycin-resistant
Enterococcus faecium. Infect
Immun 2002, 70:20410.[PubMed][Free
Full Text]
- Levin, BR & Bull, JJ: Phage therapy revisited: the population biology of
a bacterial infection and its treatment with bacteria and
antibiotics. American
Naturalist 1996, 147:881898.
- Payne, RJ, Phil, D, & Jansen,
VA: Phage therapy: the peculiar
kinetics of self-replicating pharmaceuticals. Clin Pharmacol Ther 2000, 68:22530.[PubMed][Full
Text]
- Smith, HW & Huggins, MB:
Successful treatment of experimental
Escherichia coli infections in mice using phage: its general
superiority over antibiotics. J Gen
Microbiol 1982, 128:307318.[PubMed]
- Smith, HW & Huggins, MB:
Effectivness of phages in treating
experimental Escherichia coli diarrhoea in calves, piglets and
lambs. J Gen Microbiol 1983, 129:26592675.[PubMed]
- Smith, HW, Huggins, MB, & Shaw,
KM: The control of experimental
Escherichia coli diarrhoea in calves by means of
bacteriophage. J Gen
Microbiol 1987, 133:11111126.[PubMed]
- Crow, JF & Kimura, M: An Introduction to Population Genetics
Theory. New York Harper Row
First 1971:.
- Negri, MC, Lipsitch, M, Blazquez, J,
Levin, BR, & Baquero, F: Concentration-dependent selection of small
phenotypic differences in TEM beta-lactamase-mediated antibiotic
resistance. Antimicrob Agents
Chemother 2000, 44:248591.[PubMed][Free
Full Text]
- Andersson, DI & Levin, BR:
The biological cost of antibiotic
resistance. Curr Opin
Microbiol 1999, 2:48993.[PubMed][Full
Text]
- Adams, M: Bacteriophages. New
York Interscience Publishers 1959:.
- Levin, BR, Stewart, FM, & Chao,
L: Resource – limited growth,
competition, and predation: a model and experimental studies with
bacteria and bacteriophage. American Naturalist 1977, 977:324.
- Eagle, H: The effect of the size of the inoculum and the age
of the infection on the curative dose of penicillin in
experimental infections with streptococci, pneumococci, and
Treponema pallidum. Journal of
Experimental Medicine 1949,
90:595607.
- Eagle, H, Fleischman, R, &
Musselman, AD: The bactericidal
action of penicillin in vivo: the participation of the host, and
the slow recovery of the surviving organisms. Annals of Internal Medicine 1950, 33:544571.
- Eagle, H: Experimental approach to the problem of treatment
failure with penicillin. American
Journal of Medicine 1952,
13:389399.
- Tuomanen, E, Cozens, R, Tosch, W, Zak,
O, & Tomasz, A: The rate of
killing of Escherichia coli by beta-lactam antibiotics is strictly
proportional to the rate of bacterial growth. J Gen Microbiol 1986, 132(Pt 5):1297304.[PubMed]
- Handwerger, S & Tomasz, A:
Antibiotic tolerance among clinical isolates
of bacteria. Reviews of Infectious
Diseases 1985, 7:36886.[PubMed]
- Tuomanen, E: Phenotypic tolerance: the search for beta-lactam
antibiotics that kill nongrowing bacteria. Rev Infect Dis 1986, 8(Suppl 3):S27991.[PubMed]
- Tuomanen, E & Tomasz, A:
Mechanism of phenotypic tolerance of
nongrowing pneumococci to beta-lactam antibiotics. Scand J Infect Dis Suppl 1990, 74:10212.[PubMed]
- Stent, GS: Molecular Biology of Bacterial Viruses.
San Francisco Freeman 1963:.
- Craig, W: Pharmacokinetic and experimental data on
beta-lactam antibiotics in the treatment of patients. Eur J Clin Microbiol 1984, 3:5758.[PubMed]
- Gudmundsson, S, Vogelman, B, &
Craig, WA: The in-vivo postantibiotic
effect of imipenem and other new antimicrobials. Journal of Antimicrobial Chemotherapy
1986, 18:6773.[PubMed]
- Mouton, JW, den Hollander, JG, &
Horrevorts, AM: Emergence of
antibiotic resistance amongst Pseudomonas aeruginosa isolates from
patients with cystic fibrosis. Journal of Antimicrobial Chemotherapy
1993, 31:91926.[PubMed]
- Gudmundsson, S, Einarsson, S,
Erlendsdottir, H, Moffat, J, Bayer, W, & Craig, WA:
The post-antibiotic effect of antimicrobial
combinations in a neutropenic murine thigh infection model.
Journal of Antimicrobial
Chemotherapy 1993, 31:17791.[PubMed]
- Karpman, D, Connell, H, Svensson, M,
Scheutz, F, Alm, P, & Svanborg, C: The role of lipopolysaccharide and Shiga-like
toxin in a mouse model of Escherichia coli O157:H7
infection. J Infect Dis
1997, 175:61120.[PubMed]
- Giraud, A, Matic, I, Radman, M, Fons,
M, & Taddei, F: Mutator bacteria
as a risk factor in treatment of infectious diseases. Antimicrob Agents Chemother 2002, 46:8635.[PubMed][Free
Full Text]
- Frendeus, B, Godaly, G, Hang, L,
Karpman, D, Lundstedt, AC, & Svanborg, C: Interleukin 8 receptor deficiency confers
susceptibility to acute experimental pyelonephritis and may have a
human counterpart. J Exp Med
2000, 192:88190.[PubMed][Free
Full Text]
- Korona, R, Korona, B, & Levin,
BR: Sensitivity of naturally
occurring coliphages to type I and type II restriction and
modification. Journal of General
Microbiology 1993, 139:128390.[PubMed]
- Bloch, CA & Rode, CK: Pathogenicity island evaluation in E. coli K1 by
crossing with laboratory strain K-12. Infect Immun 1996, 64:321423.[PubMed][Free
Full Text]
- Ackermann, HW: Bacteriophage taxonomy in 1987. Microbiol Sci 1987, 4:2148.[PubMed]
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Additional File
1 |
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Sensitivity of Phage Replication Assay to Growth Conditions. This
file explains some assumptions that underlie the Phage Replication
Assay (PRA), how assay conditions may violate those assumptions and
affect those values estimated, and how to minimize the effect of
violating those assumptions. A mathematical model using differential
equations is provided to evaluate the magnitudes of some of these
effects.
1471-2180-2-35-S1.pdf
Click here for file |
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