Utility of lytic bacteriophage in the treatment of
multidrug-resistant Pseudomonas aeruginosa
ORIGINAL ARTICLE
Indian Journal of Pathology and Microbiolgy
Year : 2008 | Volume : 51 | Issue : 3 | Page : 360-366
CS Vinodkumar1, Suneeta Kalsurmath2,
YF Neelagund3
1 Department of Microbiology, SS Institute of Medical Sciences
and Research Centre, Davanagere, India
2 Department of Physiology, SS Institute of Medical Sciences
and Research Centre, Davanagere, India
3 Department of Microbiology, Gulbarga University, Gulbarga,
Karnataka, India
Correspondence Address:
C S Vinodkumar
Department of Microbiology, SS Institute of Medical Sciences and Research
Centre, Vidhyanagar Post Box-1, NH-4, Bypass, Davanagere - 577 005, Karnataka
India
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/0377-4929.42511
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Drug resistance is the major cause of increase in morbidity and
mortality in neonates. One thousand six hundred forty-seven suspected
septicemic neonates were subjected for microbiological analysis over a period
of 5 years. Forty-two P. aeruginosa were isolated and the antibiogram
revealed that 28 P. aeruginosa were resistant to almost all the common
drugs used (multidrug-resistant). The emergence of antibiotic-resistant
bacterial strains is one of the most critical problems of modern medicine. As a
result, a novel and most effective approaches for treating infection caused by
multidrug-resistant bacteria are urgently required. In this context, one
intriguing approach is to use bacteriophages (viruses that kill bacteria) in
the treatment of infection caused by drug-resistant bacteria. In the present
study, the utility of lytic bacteriophages to rescue septicemic mice with
multidrug-resistant (MDR) P. aeruginosa infection was evaluated. MDR P.
aeruginosa was used to induce septicemia in mice by intraperitoneal (i.p.)
injection of 10 7 CFU. The resulting bacteremia was fatal within 48
hrs. The phage strain used in this study had lytic activity against a wide
range of clinical isolates of MDR P. aeruginosa. A single i.p. injection
of 3 × 10 9 PFU of the phage strain, administered 45 min after the
bacterial challenge, was sufficient to rescue 100% of the animals. Even when
treatment was delayed to the point where all animals were moribund,
approximately 50% of them were rescued by a single injection of this phage preparation.
The ability of this phage to rescue septicemic mice was demonstrated to be due
to the functional capabilities of the phage and not to a nonspecific immune
effect. The rescue of septicemic mice could be affected only by phage strains
able to grow in vitro on the bacterial host used to infect the animals
and when such strains are heat-inactivated, they lose their ability to rescue
the infected mice. Multidrug-resistant bacteria have opened a second window for
phage therapy. It would seem timely to begin to look afresh at this approach. A
scientific methodology can make phage therapy as a stand-alone therapy for
infections that are fully resistant to antibiotics.
Keywords: Bacteriophage, multidrug-resistant, P.
aeruginosa, mice, septicemia
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Pseudomonas aeruginosa , a Gram-negative bacterium is one of the most
important causative agents in neonatal septicemia. [1] Several outbreaks of infection caused by P.
aeruginosa isolates that are simultaneously resistant to broad-spectrum cephalosporins
and aminoglycosides have been reported. Some of these multidrug-resistant
isolates produce "Extended spectrum β-Lactamases" that are able to
hydrolyze expanded spectrum cephalosporins, aztreonam and related
oxyimino-β-lactam. [2] Colonization of such
multidrug-resistant P. aeruginosa in the hospital settings has become
very common in our region. Such colonization predisposes premature and
underweight babies to multidrug-resistant P. aeruginosa. [3] According to National Neonatal Perinatal Database
2000, the incidence of neonatal septicemia caused by P. aeruginosa is
18%. [4] The potential clinical significance of P. aeruginosa in neonatal
septicemia continues to increase as medical therapy involves more invasive
intervention procedures. Emergences of P. aeruginosa as one of the
leading nosocomial pathogens in neonatal intensive care units, especially in
high-risk babies and the widespread occurrence of multidrug-resistant among
them pose a therapeutic problem. [1]
With the rising prevalence of antibiotic-resistant bacteria, alternatives to
treatment with antibiotics are receiving increased attention. One such
alternative is the possible therapeutic use of bacteriophages - viruses that
parasitize and kill bacteria. [5] The suggestion of administering phages as
pharmaceutical agents has been mooted for more than 85 years. The relative
simplicity and economy of phage therapy should have gained much momentum and
magnitude earlier. This has not happened, probably because of a poor
understanding of mechanism of bacterial pathogenesis and of the nature of
phage-host interactions and the absence of animal models of diseases and also
due to badly designed and executed experiments and field trials which led to
failure in using phages in therapy. [5],[6],[7],[8],[9] Another reason was due to the fact that the scientists concentrated on
escalated production of newer and newer antibiotics of commercial importance.
However, the increasing incidence of multidrug-resistant bacteria and a deficit
in the development of new chemotherapeutics to counteract bacteria, [2],[4],[10] have rekindled the interest in phage
therapy. This work was carried out to investigate the possibility of use of
lytic phage in the treatment of experimental septicemic mice, which are
infected with MDR P. aeruginosa .
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Ethical clearance
The institutional ethical review board at Gulbarga University, Gulbarga
approved all the study procedures.
Bacterial strain and antibiotic sensitivity pattern
P. aeruginosa strain was isolated from the blood of a neonate with
septicemia and was designated as YFN-58 in our nomenclature. Antibiotic
susceptibility testing by Kirby-Bauer's method [10] revealed that the YFN-58 strain was
resistant to most of the commonly used drugs. Staphylococcus aureus
(ATCC no. 25923) and Escherichia More Details coli (ATCC no. 25922) were used as
control strains in antibiotic susceptibility testing.
Establishing the minimum lethal dose (MLD) of P. aeruginosa in the
mouse model
One-month-old BALB/c male mice (four per group) free from specific
pathogen, belonging to the same race, weighing 22.0 ± 1.5 g, caged singly and
maintained on a proper diet were used for infection experiments. All the mice
were housed in a pathogen-free environment within the animal-care facility at
Gulbarga University. Bacterial innocula were prepared by culturing P.
aeruginosa in brain-heart infusion overnight and centrifuging it at 2,000 ×
g for 5 min, washing, recentrifuging it twice and then resuspending it
in saline to various densities. Each group of mice received intraperitoneal
inoculation of 400µl aliquots of bacterial suspension in different densities
(10 2 -10 7 CFU). The animals were observed for 100 hrs.
Mice inoculated with bacteria were scored for their state of health on a scale
of 5 to 0, based on progressive disease states reflected by several clinical
signs. [11],[12],[13],[14] A normal and unremarkable condition was scored at 5;
slight illness, defined as lethargy and ruffled fur, was scored as 4; moderate
illness, defined as severe lethargy, ruffled fur and hunched back, was scored
as 3; severe illness, with the above signs plus exudative accumulation around
partially closed eyes, was scored as 2; a moribund state was scored as 1; and
death was scored as 0. Scores were determined by two independent observers.
Isolation and purification of phage strains for P. aeruginosa
The P. aeruginosa phage was isolated from raw sewage at a municipal
sewage treatment plant, Gulbarga by the method of Smith and Huggins. [8] Sewage water (50 ml) was collected in a sterile
conical flask and treated with a few drops of chloroform. To this, an equal
volume of sterile nutrient broth and 1 ml of the 24-hour-old broth culture of P.
aeruginosa YFN-58 were added. The sample inoculated with bacterial
pathogens was incubated at 37°C for 12-24 hrs in shaker water bath. After 12-24
hrs, the lysate was shaken with few drops of chloroform for about 10 min,
centrifuged at 10,000 rpm for 10 min and the supernatant was filtered through
0.22 µ pore size acrodisk membrane filters (PALL, German Laboratory) to remove
the bacteria, [11] and subjected to plaque-forming unit (PFU) assay
using the double-layer agar method described by Smith and Huggins. [8] The phage was denoted as CSV-31.
In vitro confirmation of bacteriophage activity on P. aeruginosa
The bacterial lawn was prepared on nutrient agar plates employing 1.0 ml of 24
hrs P. aeruginosa YFN-58 culture by flooding and draining out the
excess. Wells were dug into the agar by employing a sterile cork borer and the
20µl phage suspension (3 × 10 9 PFU/ml) was loaded into each of the
well. Sterile distilled water served as the control. The plates were incubated
at 37°C for 24 hrs. Thereafter, the zone of inhibition, if any, was recorded.
[12],[13]
Treatment with phage
The efficacy of phage therapy was evaluated in two separate experiments
using MDR P. aeruginosa bacteremic mouse model. The first experiment was
to examine the effect of phage dose on the ability of phage to rescue mice from
MDR P. aeruginosa bacteremia. In the second study, on the outcome of
delaying treatment for various periods. In the dose-ranging study, five groups
of mice (four mice in each) were challenged by i.p. injection of the MLD of
YFN-58. Each of these groups was treated with a single injection of phage
CSV-31 administered i.p. 45 min after the bacterial challenge at 3 × 10 9
, 3 × 10 8 , 3 × 10 4 and 0 PFU. As an additional
control, a sixth group (two mice) was not challenged with bacteria, receiving
only the injection of phage (at the highest dose). The state of health of these
animals was monitored for 20 days. [14]
In the delayed-treatment study, treatment (a single injection of phage at the
highest dose) was initiated at 5, 8, 12, 16 and 24 hrs after the bacterial
challenge with the MLD P. aeruginosa YFN-58. The state of health
of these animals was also monitored for 20 days. [10],[13],[15]
Effects of heat-inactivated phage
A sample of phage with a titer of 3 × 10 9 PFU/ml was
heat-inactivated by incubation at 80°C. Phage that had been heated for a total
of 20 min, at which time no viable phage was detectable, was used to determine
whether phage rescue mice with MDR Pseudomonas bacteremia requires
functional phage or whether the rescue might be associated with nonspecific
immune activation. [16],[17] The mice in this study were divided into two groups
of four each. All of the mice were challenged by i.p. injection of 3 × 10
9 PFU of phage CSV-31, 45 min after the bacterial challenge. The second
group was treated with i.p. injection with 3 × 10 9 PFU of
heat-inactivated phage particles 45 min after the bacterial challenge.
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Antibiotic susceptibility testing
Antibiotic sensitivity testing revealed that out of 42 P. aeruginosa
isolated from neonatal septicemia, 28 isolates were multidrug-resistant. All
these 28 isolates were extended spectrum β lactamase producers.
Lethality of MDR P. aeruginosa bacteremia
As seen in [Figure 1], all mice inoculated i.p. with MLD 10 7
CFU of the clinical isolate MDR P. aeruginosa YFN-58 died within 48 hrs.
Phage strain antibacterial activity
Of the phage strains isolated in the present study, phage CSV-31 was found
to form plaques on 66% of the MDR Pseudomonas clinical isolates and
inhibited bacterial growth of an additional 8% of the strains, thus exhibiting
an antibacterial effect against 74% of the strains in our collection.
Ability of the phage preparation to rescue mice from MDR Pseudomonas
bacteremia
A single dose of phage CSV-31 was administered i.p. 45 min after the
challenge with the MLD of bacteria. By 24 hrs, a dose effect on the state of
health of the infected animals was clearly visible. At higher doses of phage,
100% of the animals survived and only minimal signs of illness (mild lethargy)
were seen (in the first 24 hrs). As the phage dose decreased, the animals
became critically ill, with survival rates of 40% and 60%, respectively, at day
6 and beyond [Figure 2]. All the mice that were alive and healthy at day 6
remained so for an additional 20 days, at which time the experiment was
terminated.
Effect of delay in treatment on the ability of the phage preparation to
rescue mice from MDR Pseudomonas bacteremia
In this experiment, the MLD of MDR Pseudomonas strain YFN-58 was
injected i.p. to induce fatal bacteremia. At various intervals thereafter,
ranging from 2 to 24 hrs, the mice received a single i.p. injection of the high
dose (3 × 10 9 PFU) of phage CSV-31. The results of treatment at the
time points are illustrated in [Figure 3]. The state of health of these mice was monitored for
20 days following bacterial infection. The experiments demonstrate that a
single injection of phage can rescue 100% of the animals, even when treatment
is delayed until 5 hrs after lethal bacterial challenge and if treatment is
delayed beyond that point, morbidity increases and mortality begins to appear.
However, even with delay of 18 and 24 hrs, at which point all the mice are
moribund, approximately 50% of the animals are rescued and recovered
completely.
Effects of heat-inactivated phage
An experiment was performed to determine whether phage rescue of mice with
multidrug-resistant Pseudomonas bacteremia requires phage that can grow
on the bacterial host or whether phage rescue might be associated with a
nonspecific immune activation response. Heating at 80°C for 5 min decreased the
phage titer by 1000-fold and no viable phage was detected after heating for 15
to 20 min. As illustrated in [Figure 4], only mice inoculated with plaque-forming phage had
enhanced survival, with 75% survival at 4 days and with 10% of the mice
injected with heat-inactivated phage surviving.
Rescue is associated with a significant decrease in bacterial titer
In a similar experiment, blood was obtained by cardiac puncture during a
rescue experiment in order to compare bacterial titers from two groups of four
mice each, 20 hrs after i.p. inoculation with 3 × 10 7 CFU of
YFN-58. Forty-five minutes after the bacterial inoculation of the first group,
250µl of PBS was injected i.p. as a control. The bacterial titer in the blood
20 hrs after bacterial inoculation for this group of mice was 1.4 × 10 7
± 1.2 × 10 7 CFU/ml (Mean ± standard error). Forty-five minutes
after bacterial inoculation of the second group, the mice were injected i.p.
with 9 × 10 9 PFU of phage CSV-31. The mean bacterial titer in the
blood 20 hrs after bacterial inoculation for this group was 6.4 × 10 4 ±
4.8 × 10 4 CFU/ml. Phage therapy thus resulted in a 300-fold
decrease (compared to control group) in blood bacterial titers at 20 hrs. This
reduction in bacterial titers was reflected in the lower morbidity and
mortality observed in the treated group. Whereas at the 20-hr time point the
control group had a median health score of 2 (severe illness), with scores
ranging from 0 (one death) to 4, in contrast the phage-treated group had a
median health score of 3 (moderate illness), with no deaths and scores ranging
from 2 to 5. In both groups, there was a correlation between the state of
health of the mice and the concentration of bacteria in the blood. Mice with
bacterial titers of 10 6 CFU/ml or more had a score of 1 (moribund
condition), while titers in the range of 10 5 to 10 4
CFU/ml resulted in a score of 2. Mice with 10 3 CFU of bacteria/ml
in their blood displayed a score of 3 and mice with titers of 10 2
CFU/ml or less had scores of 4 to 5 (mild illness or no signs of illness,
respectively).
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Septicemia is a leading cause of morbidity and mortality among neonates in
developing countries. But, the indiscriminate overuse of antibiotics for the
treatment of septicemia has triggered an increase in the development of
multidrug-resistant "Super bugs" and has been posing serious
problems. [1],[4] Therefore, in the present investigation, an attempt
is made to develop an alternative to conventional drugs. One possible option is
to use bacteriophages as antimicrobial agents; the bacteriophages, which are
highly specific for the target bacteria, without affecting the normal
microflora and are effective against multiple drug-resistant bacteria.
Although phages were discovered nearly a century ago, Western medicine's
interest in them as therapeutic agents was relatively short-lived, in part
because of the eventual discovery and immediate success of antibiotics and in
part because of the highly empirical and counterproductive approach that had
been used by phage practitioners in the early era. In the modern era (1980s and
1990s), some rigorously controlled animal experiments have been conducted
(Smith Soothill), [8],[16],[17],[18] but the clinical
reports in this same era have been of an anecdotal nature rather than
describing controlled studies. [19],[20],[21] The experiments in the present study represent
solutions to many of the problems that hindered the prior applications of phage
therapy. For example, the relatively narrow host range of most phages which
caused many of the early attempts at phage therapy to fail can be overcome by
isolating phages that have a broad host range within the species being
targeted. For example, phage CSV-31 forms plaques on more than 66% of the MDR Pseudomonas
isolates tested and inhibits growth of an additional 8%, thus providing an
antibacterial effect against more than 74% of approximately 46 clinical
isolates of MDR Pseudomonas isolated from neonatal septicemic cases. The
inhibition of bacterial growth in strains that the phage could not form plaques
(in 26% of clinical isolates) is most likely due to partial expression of the
phage genome, [19],[20] sufficient for
killing but not enough for phage production to a level necessary for plaque
formation.
With the emergence of antibiotic-resistant bacteria, such as MDR Klebsiella
, methicillin-resistant Staphylococcus aureus, E. coli, P. aeruginosa and
vancomycin-resistant Enterococci , there is a need to explore the
potential therapeutic applications of bacteriophages. [14],[15],[21] That exploration is facilitated by knowledge gained
during the development of the science of molecular biology about the mechanisms
of phage interactions with bacteria. This knowledge, coupled with rigorously
controlled animal models and clinical trials, should permit the rational
development of phage therapy. In this regard, the inocula of bacteria used in
the animal experiments described in this study are higher than those normally
encountered in clinical situations. However, the ability of phage to rescue
animals infected with these high titers of bacteria is especially convincing of
the potential of phage as an antibacterial therapeutic agent. Given the
encouraging results of these initial experiments, it may be useful to develop
more sophisticated experiments that portray normal clinical situations.
The results obtained in the present study are encouraging: a single injection
of either of the two dosages of phage used in this study (10 9 and
10 8 PFU) rescued 100% of MDR Pseudomonas septicemic mice,
even if treatment was delayed up to 5 hrs. Moreover, even when phage treatment
(with the highest dose) was delayed until all animals were moribund,
approximately half the animals were rescued and went on to recover completely.
The ability of phage to rescue bacteremic animals was a phage function and not
nonspecific immune response activation, since rescue was observed with
heat-inactivated phage.
Each of the above experiments independently demonstrates the requirement for
active phage, that is, phage that are capable of growing in and lysing the infecting
bacterial strain, for the rescue of animals that are bacteremic with that
bacterial strain. This conclusion was further strengthened by observations
concerning the fate of the bacteria during infections. In these experiments, we
observe a 300-fold decrease in blood bacterial titers in mice treated with
phage therapy when compared with blood bacterial titers from the PBS-treated
control mice 20 hrs after initiation of the experiment.
Phage CSV-31 persisted long enough in the tissues to be effective when
administered to the mice a day or two before challenge with the P.
aeruginosa . This was also reported previously with chicken [20],[21] and suggests that
there are possibilities for prophylaxis. More interestingly, it was possible to
delay administration of the phage until signs of disease were evident in the
mice and yet retain considerable protection, suggesting that acute infections
might also be amenable to phage treatment.
Pioneering study by Shankar Adhya indicated that bacteriophage therapy could be
used in gastrointestinal colonization with vancomycin-resistant Enterococcus
infection. [11] A single i.p. injection of the phage strain was
sufficient to rescue 100% of the animals. Even in the present study, we could
demonstrate that viable phage were able to rescue 75% of the septicemic mice.
We focused our efforts on MDR Pseudomonas because we anticipated that
positive results would demonstrate the potential of this form of therapy in
situations where few alternatives are available today. It is tempting to
advocate research investigations into many bacterial infections for which
animal models are available and for which phages may be isolated. The potential
of phage therapy has been the subject of several recent reviews, [15],[16],[17],[18] and the present study reinforces the view
that this therapy is worth exploring.
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The authors would like to thank the Department of Pathology, NIMHANS, the Department
of Zoology and the Department of Microbiology, Gulbarga University, Gulbarga
for the facilities.
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Correspondence Address:
C S Vinodkumar
Department of Microbiology, SS Institute of Medical Sciences and Research
Centre, Vidhyanagar Post Box-1, NH-4, Bypass, Davanagere - 577 005, Karnataka
India
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/0377-4929.42511
