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REVIEW OF LITERATURE
17
REVIEW OF LITERATURE
2.1 BACTERIOPHAGE THERAPY
Prior to the discovery and widespread use of antibiotics, it was
suggested that bacterial infections could be prevented and/or treated by the
administration of bacteriophages (viruses attacking bacteria). Although the
early clinical studies with bacteriophages were not rigorously pursued in the
United States and Western Europe, phages continued to be utilized in the
former Soviet Union and Eastern Europe. Ernest Hankin, a British
bacteriologist, reported in 1896 on the presence of marked antibacterial
activity (against Vibrio cholerae) which he observed in the waters of the
Ganges and Jamuna rivers in India, and he suggested that an unidentified
substance (which passed through fine porcelain filters and was heat labile)
was responsible for this phenomenon and for limiting the spread of cholera
epidemics. Two years later, the Russian bacteriologist Gamaleya observed a
similar phenomenon while working with Bacillus subtilis, and the observations
of several other investigators are also thought to have been related to the
bacteriophage phenomenon.
However, non of these investigators further
explored their findings until Frederick Twort, a medically trained bacteriologist
from England and Felix d’Herella, a French Canadian bacteriologist
reintroduced the subject almost 20 years after Hankin’s observation by
reporting a similar phenomenon and advancing the hypothesis that it may
have been due to a virus (Sulakvelidze et al., 2001).
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Biomedical technology today is very different from what it was in the
early days of phage therapy research, and our understanding of biological
properties of phages and the basic mechanisms of phage-bacterial host
interaction has improved dramatically since the days of early therapeutic uses
of bacteriophages (Sulkvelidze and Kutter, 2004). The concept of phage
therapy to treat bacterial infections was born with the discovery of the
bacteriophage almost a century ago. After a chequired history, its current
renaissance is fueled by the dangerous appearance of antibiotic-resistant
bacteria on a global scale.
As a mark of this renewed interest, the
unanswered problems of phage therapy are now being addressed, especially
for human use (Duckworth and Gulig, 2002).
2.1.1 Development
From the time Felix d’Herelle discovered bacteriophage he was
interested in their relationship agents.
He systematically investigated the
nature of bacteriophages and explored their ability to function as therapeutic
agents d’Herellel (1917). The studies on treatment of bacterial dysentery by
using phages were conducted by Felix d’Herelle at Paris hospital in the
summers of 1919 (Summers, 1999). The major milestones in the
development of phage therapy and important studies on human phage
therapy conducted by various researchers throughout the world are recorded
in brief as under.
Spence and McKinley (1924), Bacterial dysentery: Phages were
administrated orally to 20 patients infected with Shiga (9 patients) and Flexner
(11 patients) bacillus. Twelve patients not treated with bacteriophage, but
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treated with the conventional methods, served as control group during the
study. 30% less mortality was reported in phage-treated patients compared
to phage-untreated patients.
d’Herelle (1925), Plague: 4 plague patients recovered dramatically
after injecting bacteriophages into their bubos.
This is the first published
report of the use of phage therapy against plague. The success outcome was
largely responsible for the subsequent initiation of large-scale studies of the
efficacy of phages against plague and cholera in India.
d’Herelle et al. (1928), Enteric infections: Orogastric administration of
phages dramatically reduced the cholera-associated fatality rate from
27-30% to Zero, in the Campbell hospital in Calcutta. During field trials, the
prophylactic/therapeutic administration of phages to 74 patients in Punjabi
villages reduced the mortality rate to 8%, compared to 63% among the
124 patients not treated with phages.
Burnet et al. (1930), Dysentery: Anti-dysentery phages were used for
prophylaxis/treatment of dysentery.
Babalova et al. (1968), Dysentery: 17,044 children were treated with
phage preparations vx. 13,725 children in control groups. Based on clinical
diagnosis, the incidence of dysentery was 3.8–fold less than that occurring in
the control, phage untreated group.
Isoliani et al. (1980), Lung infections: Phages were successfully used,
together with antibiotics, to treat lung and pleural infections in
patients.
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Meladze et al. (1982), Lung infections: Phages were used to treat
223 patients with infections of the lung parenchyma and pleura, and the
results were compared to those of 117 patients treated with antibiotics.
Full recovery was observed in 82% of the patients in the phage-treated group,
as opposed to 64% of the patients in the antibiotic treated group.
El-Tahan et al. (1983), Burnt infections: Reported the successful
bacteriophage therapy in burn wound sepsis caused by P. aeruginosa and
the recovery in almost all the patients was observed.
Martynova et al. (1984), Opportunistic infections: S. aureus and
P. aeruginosa specific phages were used prophylactically (in forms of mouth
rinses) 2 times/day for 3-5 days in 27 patients. Control group consisted of
10 healthy individuals. Phage treatment was associated with normalization of
microflora in infected sites, and it also stimulated the production of secretory
immunoglobulins (IgA in particular).
Cislo et al. (1987), Skin Ulcers: 31 patients with chronically infected
skin ulcers were treated orally and locally with phages. The success rate was
74%.
Minor side effects (eczema, pain, or vomiting) were observed in
6 patients.
Kochetkova et al. (1989), Post-operative infections: 131 cancer
patients with post-surgical wound infections participated in the study.
Of these, 65 patients received phages, alone or in combination with
antibiotics, and the rest received antibiotics. Phage treatment was successful
in 82% of the cases, and antibiotic treatment was successful in 61% of the
cases.
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Abul-Hassan et al. (1990), Burnt infections: Phage therapy was used
to control P. aeruginosa infection in 30 cases of resistant infections in patients
with burn areas ranging from 10-25%. Post-phage cultures showed absence
of organisms in 12 patients and their presence in 18. Satisfactory graft take
was present in 18 cases only and inferior take in 12 cases.
Sakandelidze (1991), Infectious allergoses: 1,380 patients with
infectious allergoses were treated with phages
(360 patients), antibiotics
(404 patients), or a combination of phages and antibiotics (576 patients).
Clinical improvement was observed in 86, 48 and 83% of the cases,
respectively.
Miliutina and Vorotyntseva (1993), Salmonellosis: The efficacy of
treating salmonellosis using phages and a combination of phages and
antibiotics was examined. The combination of phages and antibiotics was
reported to be effective in treating cases where antibiotics alone were
ineffective.
Kwarcinski et al. (1994), E. coli infections: Recurrent subphrenic
abscess (After stomach resection) caused by an antibiotic resistant strain of E.
coli was successfully treated with phages.
Perepanova et al. (1995), Urogenital infections:
Adapted phages
were used to treat acute and chronic urogenital inflammation in 46 patients.
The efficacy of phage treatment was 92% (marked clinical improvements) and
84% (bacteriological clearance).
Weber-Dabrowska et al. (2001), Cancer: 20 Cancer patients refractory
to treatment with commonly available antibiotics were treated by oral
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administration of specific phages (3 doses/day, for 2 to 9 weeks).
Some
patients were also treated by local administration of phages. Complete
healing of the local lesions and termination of the supurative process was
reported for all phage-treated patients. Side effects were not observed in any
of the patients subjected to phage treatment.
Markoishvili et al. (2002), Wound infections: The wounds healed
completely in 67 (70%) of 96 patients whose wounds were covered with a
phage-containing biodegradable matrix. In 22 cases in which microbiological
data were available, healing was associated with the concomitant elimination
of, or a reduction in, specific pathogenic bacteria in the wounds.
Paisano et al. (2004), Dental Infection: In vitro antimicrobial effect of
bacteriophages on human infected dentin was studied. Results revealed high
efficacy of bacteriophages on human dentin infected with Enterococcus
faecalis ATCC 29219.
2.1.2 Prospects
Infectious disease experts have warned that there is now a compelling
need to develop totally new classes of antibacterial agents, ones that cannot
be resisted by the same genes that render bacteria resistant to antibiotics.
Phage therapy represents such a new class. The impediments like bacterial
debris in the preparations, rapid clearance in the body, etc. can be overcome,
frezing up the phages so that their attributes such as, exponential growth, and
the ability to mutate against resistant bacteria can be used to great advantage.
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The three additional attributes of phages as their future prospects
according to Carlton (1999) are as mentioned below.
Host specificity: While the host specificity is somewhat of a drawback
(requiring a match up of phage to bacterial target, and/or the development of
highly multivalent phages), it also offers the great advantage that the phages
will not kill other species of bacteria. Thus, e.g., phage therapy is not likely to
kill off the healthy flora of the intestines, lungs or urogential tract, and it is
therefore unlikely to provoke the illnesses and deaths seen when antibiotics
cause overgrowth of pathogens (such as Clostridia difficile and Candida
albincans).
Genetic engineering: It is possible to genetically engineer phages to express
new traits of potential value. In so doing, scientists will have to deal with the
legitimate
concerns
of
regulatory
agencies
concerning
recombinant
organisms. The regulatory obstacles may be well worth the price, given the
powerful engineering tools that are currently available.
Ideal candidates for co-therapy with antibiotics: If a given bacterium
acquires resistance to a phage (e.g., by a mutation in the receptor site or in
the endonucelase enzymes), the mutation is likely to “teach” the bacterium to
resist the antibiotics (which do not target those structures). Similarly, if a
given bacterium acquires resistance to an antibiotic (e.g., by a mutation in the
reflux pump or in the ribosomal subunits), that mutation is not likely to “teach”
the bacterium to resist the phage (which does not target those structures).
Thus, if the bacterium is exposed to both agents, the odds are remote that
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any resistance genes it starts to express (or acquires anew) will enable it to
survive. There are reports that bacteria tend to mutate against antibiotics
once in every 106 divisions, while they tend to mutate against phages once in
every 107 divisions. Therefore the odds of a given bacterium mutating against
a phage and an antibiotic at the same time would be the product of 106x107,
meaning it would likely take 1013 bacterial divisions for such a double mutation
to occur.
Given that low probability, the co-administration of phages and
antibiotics may help prevent the emergence of bacterial resistance to
antibiotics, thereby greatly prolonging their clinical usefulness (and vice
versa). Just as multiple classes of anti-HIV medications are administered to
AIDS patients, to prevent the emergence of resistant strains of that virus, so it
is that co-therapy with phages and antibiotics may also prove to be of great
clinical value. From a clinical standpoint, phages appear to be very safe.
This is not surprising, that humans are exposed to phages from birth (and,
possibly, even in utero). The abundance of phages in the environment – and
the continuous exposure of humans to them – explains the extremely good
tolerance of the human organisms to phages. These observations strongly
suggest that phage therapy may provide one of the safest as well as most
environmentally friendly methods currently available for prophylaxis and
treatment of bacterial infections. However, in order not to compromise the
safe use of therapeutic phage preparations, they should be produced, purified,
characterized, and tested using current, state of the art biotechnology. This
approach will help ensure the consistency and safety of therapeutic phage
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preparations, and it is also likely to increase their therapeutic and prophylactic
potential.
The important advantages of using phages over antibiotics attributing
to the major prospects of phage therapy according to Ahmad (2002) are as
follows.
Phages
Antibiotics
Highly specific to the targeted infection
Less specific hence all sensitive bacteria
including normal flora destroyed
Application intervals may be as long as Required
once every 7 days
usually
at
short
intervals
(multiple applications per day)
Once applied it can continue working as Have
long as the host (infection) persists
a
limited
life
hence
regular
administration required
Grow and destroy at the site of infections Usually metabolized and distributed all
hence available where most needed
over the body
Exist for virtually all known bacteria
Some bacterial species are notoriously
resistant to several antibiotics
Can be dispersed in any media
Some may be insoluble in water
Low production cost
Relatively more expensive
Exponential growth leading to continuous No such increase
fight against infection
Natural abundance
Limited supply
Multiple resistance highly unlikely
More common due to MDR plasmids
Minimal resistance (no plasmid-borne Can be high due to plasmid-borne
resistance is known)
resistance
Shelf life can be long if stored appropriately Some have short shelf life
No disruption to normal flora
Disturbance to normal flora leading to
side effects such as secondary infection
by yeast
Side effects including serious reactions
No serious side effects known
known
Phage-resistance bacteria remain sensitive Resistance is not limited to target bacteria
to other phages with different target sites
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2.1.3 Limitations
The
discovery
of
the
bacteriophage
or
phage
is
somewhat
controversial. The consensus is that 20 years after initial observations of
unfilterable, heat labile agents with activity against – Vibrio cholerae (Ernest,
1986), it was discovered by Twort in 1915, who made similar observations
and who hypothesized this to be due to a virus, and independently by
d’Herelle in 1917 (d’Herelle, 1917; Topley and Wilson, 1936). Since it was
realized that these bacterial viruses destroy their bacterial host while
remaining harmless to humans, it has been the dream of researchers to use
phages to treat bacterial infections. One of the most important factors that
contributed to the decline of interest in phage therapy in the western world
was a credibility problem. A paucity of appropriately conducted studies and
the lack of well-established and standardized testing protocols interfered with
rigorously documenting the value of phage therapy.
In addition, several
companies started producing phages commercially and, in their eagerness to
boost profits, some made exaggerated claims concerning the effectiveness of
their products. Many productions – related problems also complicated early
phage therapy research. Before the availability of spray-dryers, freeze-dryers,
refrigerators and freezers, the viability of phage preparations was a concern;
phage titers could rapidly decline and render the preparations ineffective.
Thus, several “stabilizers” and “preservatives” were used in an attempt to
increase the viability of phages.
However, in the absence of a good
understanding of the biological nature of phages and their stability to various
physical and chemical agents, many of the ingredients added to prolong
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phage shelf-life (e.g., phenol, which was included in some of the early
therapeutic phage preparations) actually had a detrimental effect on phage
viability and were toxic for humans.
2.2 PSEUDOMONAS AERUGINOSA
Pseudomonas aeruginosa was first detected by Gessard in 1982 from
a blue pus. Until recently, only P. aeruginosa is considered as important
human pathogen, from among numerous species of Pseudomonads
(Greenwood, 1998). P. aeruginosa is an aerobic, motile, Gram –ve rod. The
production of colourful water-soluble pigments, particularly bluish green
pycocynanin
and
yellowish
green
pyoverdin
are
the
outstanding
bacteriological features of this human pathogen. It is the epitome of an
opportunistic pathogen of humans and also demonstrates the most consistent
resistance to antimicrobials of all the human pathogens (Ahmad, 2002). It is
being considered as dangerous and dreaded human pathogen because of its
notorious resistance to wide range of antibiotics.
2.2.1 Pathogenicity
P. aeruginosa is an opportunistic human pathogen, it is one of
particular virulence. The pathogen generally requires a significant break in
first line defenses (such as wound) or a route past them (such as a
contaminated solution or intratracheal tube) to initiate infection (Kenneth and
Stanley, 2002). The major infections of P. aeruginosa are both invasive and
toxigenic. However, the ultimate infection may be seen under three distinct
phases: colonization, invasion and dissemination. The disease process may
stop at any stage. Particular bacterial determinants of virulence mediate each
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of these stages and are ultimately responsible for the clinical symptoms that
accompany the disease.
Colonization:
The fimbriae of Pseudomonas will adhere to the epithelial
cells of the upper respiratory tract and, by inference, to other epithelial cells
as well. These adhesions appear to bind to specific galactose or mannose or
sialic acid receptors on epithelial cells. Colonization of the respiratory tract by
Pseudomonas requires fimbrial adherence and may be aided by production of
a protease enzyme that degrades fibronectin in order to expose the
underlying fibrial receptors on the epithelial cell surface. Tissue injury may
also play a role in colonization of the respiratory tract since P. aeruginosa will
adhere to tracheal epithelial cells of mice infected with influenza virus but not
to normal tracheal epithelium. Besides pili and the mucoid polysaccharide,
there are possibly two other cell surface adhesins utilized by Pseudomonas to
colonize the respiratory epithelium or mucin.
Invasion: The ability of P. aeruginosa to invade tissue depends upon its
resistance to phagocytosis and the host immune defenses, and the
extracellular enzymes and toxins that break down physical barriers and
otherwise contribute to bacterial invasion. The bacterial capsule or slim layer
effectively protects cells from opsonization by antibodies, complement
deposition, and phagocyte engulfment.
Two extracellular proteases have been associated with virulence that
exerts their activity at the invasive stage.
Dissemination: Blood stream invasion and dissemination of Pseudomonas
from local sites of infection is probably mediated by the same cell associated
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and extracellular products responsible for the localized disease, although it is
not entirely clear how the bacterium produces systemic illness. P. aeruginosa
is resistant to phagocytosis and the serum bactericidal response due to its
mucoid capsule and possibly lipo-polysaccharides.
The lipid A moiety of
Pseudomonas
mediates
lipo-polysaccharides
(endotoxin)
pathologic aspects of Gram-negative septicemia.
the
usual
It is also reasonable to
assume that Pseudomonas Exotoxin A exerts some pathologic activity during
the dissemination stage.
P. aeruginosa produces two extracellular protein toxins, Exoenzyme S
and Exotoxin A. Exoenzyme S is probably an exotoxin. It has the
characteristic subunit structure of the A-component of a bacterial toxin, and it
has Adinosine di-phosphate ribosylating activity (for a variety of eukaryotic
proteins) characteristic of exotoxins. Exoenzyme S is produced by bacteria
growing in burned tissue and may be detected in the blood. It has been
suggested that exoenzyme S may act to impair the function of phagocytic
cells in the bloodstream and internal organs to prepare for invasion by
P. aeruginosa.
2.2 .2 Pathogenesis
P. aeruginosa is a physiologically versatile organism, widely distributed
in soil, water, sewage, the mammalian gut and plants. P. aeruginosa is
susceptible to drying but will survive for many months in water at ambient
temperature. Simple nutritional requirements and the ability to metabolize a
variety of organic substances may enable P. aeruginosa to survive and
multiply in fluids and moist environments found in hospital wards. Infection
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with
P.
aeruginosa
is
generally
restricted
to
hospitalized
patients.
Predisposing factors for nosocomial infections include metabolic, hematologic,
and malignant diseases. Hospital-acquired infections occur in patients who
have had prior instrumentation or manipulative procedures such as urethral
catheterizations, tracheotomies, lumbar punctures, and intravenous infusions
of medications and fluids.
Patients become susceptible to P.aeruginosa
infections after prolonged treatment with immunosuppressive agents,
corticosteroids, antimetabolities, antibiotics, and radiation. Hospitalized
patients may acquire the bacteria from common environmental sources by
contact with human or inanimate vectors.
The major infections caused by P. aeruginosa are as follows (Keneeth,
2002).
Respiratory infections: Respiratory infections caused by P. aeruginosa
occur almost exclusively in individuals with a compromised lower respiratory
tract or a compromised systemic defense mechanism. Primary pneumonia
occurs in patients with chronic lung disease and congestive heart failure.
Bacteremic pneumonia commonly occurs in neutropenic cancer patients
undergoing chemotherapy.
Lower respiratory tract colonization of cystic
fibrosis patients by mucoid strains of P. aeruginosa is common and difficult, if
not impossible to treat.
Urinary tract infections: Urinary
tract
infections
(UTI)
caused
by
P. aeurginosa are usually hospital-acquired and related to urinary tract
catheterization, instrumentation or surgery. P. aeruginosa is the third leading
cause of hospital-acquired UTIs, accounting for about 12 percent of all
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infections of this type. The bacterium appears to be among the most adherent
of common urinary pathogens to the bladder uroepithelium. The urinary tract
infection can occur via an ascending or descending route. In addition,
Pseudomonas can invade the bloodstream from the urinary tract, and this is
the source of nearly 40 percent of Pseudomonas bacteremias.
Wound and burnt infections: P. aeruginosa can cause a variety of skin
infections, both localized and diffuse. The common predisposing factors are
breakdown of the integument which may result from burns, trauma or
dermatitis; high moisture conditions such as those found in the ear of
swimmers and the toe webs of athletes and combat troops, in the perineal
region and under diapers of infants, and on the skin of whirlpool and hot tub
users; neutropenia; and AIDS, Pseudomonas has also been implicated in
folliculitis and unmanageable forms of acne vulgaris.
Ear infections including external otitis: P. aeruginosa is the predominant
bacterial pathogen in some cases of external otitis including “swimmer’s ear”.
The bacterium is infrequently found in the normal ear, but often inhabits the
external auditory canal in association with injury, maceration, inflammation, or
simply wet and humid conditions.
Bacteremia: P. aeruginosa causes bacteremia primarily in immunocompromised patients.
Predisposing conditions include hematologic
malignancies, immunodeficiency relating to AIDS, neutropenia, diabetes
mellitus and severe burns. Most Pseudomonas bacteremia is acquired in
hospitals and nursing homes. Pseudomonas accounts for about 25 percent
of all hospital acquired Gram–negative bacteremias.
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Gastrointestinal infections: P. aeruginosa can produce disease in any part
of the gastrointestinal tract from the oropharynx to the rectum. As in other
forms of Pseudomonas disease, those involving the gastrointestinal tract
occur primarily in immunocompromised individuals. The organism has been
implicated in perirectal infections, pediatric diarrhea, typical gastroenteritis,
and necrotizing enterocolitis. The GI tract is also an important portal of entry
in Pseudomonas septicemia.
2.2.3 Drug resistance
Clinically significant infections with P. aeruginosa should not be treated
with single drug therapy, because the success rate is low with such therapy
and because the bacteria can rapidly develop resistance when single drugs
are employed. A penicillin active against P. aeruginosa- ticarcillin, mezlocillin,
and piperacillin – is used in combination with an aminoglycoside, usually
gentamicin, tobramycin, or amikacin. Other drugs active against P.aeruginosa
include
aztreonam,
imipenem
and
the
newer
quinolones,
including
ciprofloxacin. Of the newer quinolones, including ciprofloxacin, ceftazidime
and cefoperazone are active against P. aeruginosa; ceftazidime is used in
primary therapy of P. aeruginosa infections (Norrby et al., 1993).
To a considerable extent, the intrinsic resistance of P. aeruginosa is
due to outer membrane porins that restrict the entry of antimicrobials to the
periplasmic space. The bacterium is naturally resistant to many antibiotics
due
to
the
permeability
lipopolysaccharide.
barrier
afforded
by
its
outer
membrane
Since its natural habitat is the soil, living in association
with the bacilli, actinomycetes and molds, it has developed resistance to a
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variety of naturally occurring antibiotics. Moreover, Pseudomonas maintains
antibiotic resistance plasmids and it is able to transfer these genes by means
of transduction and conjugation (Farjadian et al., 1996 and Kenneth, 2002).
P. aeruginosa strains are regularly resistant to pencillin, ampicillin,
cephalothin, tetracycline, chloramphenicol, sulfonamides and the earlier
aminoglycoisdes (streptomycin, kanamycin). Much effort has been directed
towards the development of antimicrobic with anti-Pseudomonas activity. The
aminoglycosides, gentamicin, tobramycin, and amikacin, are all active against
most strains despite the presence of mutational and plasmid mediated
resistance. Carbenicillin and ticarcillin are active and can be given in high
doses, but plasmid-mediated resistance and permeability mutations occur
more frequently than with the aminoglycoides. A primary feature of the third
generation cephalosporins (ceftazidime), carbapenems (imipenem), or
monobactams
(azthreonam)
is
their
activity
against
Pseudomonas.
In general, more serious systemic P. aeruginosa infections are usually treated
with a combination of an anti-Pseudomonas beta-lactam antimicrobic and an
aminoglycoside, particularly in neutropenic patients. Ciprofloxacin is also
used in treatment of such cases.
In all instances susceptibility must be
confirmed by in vitro susceptibility tests.
2.3 BACTERIOPHAGE
Bacterial viruses or bacteriophages, frequently called “phages”, were
discovered twice at the beginning of the 20th century. Frederick William Twort,
a British pathologist in London, described in 1915 the glassy transformation of
“Micrococcus” colonies by a transmissible agent.
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He proposed
several explanations, one of which was that the agent was viral in nature.
Felix Hubert d’Herelle, a French Canadian then working at the Pasture
Institute of Paris, observed the lysis of Shigella cultures in broth and
described it in 1917. Twort did not pursue his discovery, but attempted for
decades to propagate vertebrate viruses on inert media. d’Herelle, on the
contrary, clearly recognized the viral nature of his agent and devoted the rest
of his scientific life to it. He coined the term “Bacteriophage”, devised several
techniques still in use, postulated the intracellular multiplication of viruses,
and introduced phage therapy of infectious diseases (d’Herelle, 1917). The
viral nature of bacteriophages, contested for a time, was definitely recognized
in 1940 after the advent of the electron microscope (Pfankuch and Kausche,
1940).
2.3.1 Biology
The study of phages begins with the work of Max Delbruck in the late
1930’s, Nobel Laureate Delbruck, originally a physicist, began studying
phages as genetic and biochemical experimental systems.
This work led
others to focus their studies on the structure biology and assembly of phages.
Life cycle: Bacteriophages undergo two possible life cycles. These are the
lytic (or virulent) and lysogenic. Lytic phage multiply vegetatively and kill the
host cell at the end of the growth cycle. Temperate phages which undergo
the lysogenic cycle as well as multiplying vegetatively can also persist in a
lysogenic state, whereby the phage genotype can exist indefinitely by being
inserted in the bacterial chromosome (known as the prophage state) (Karl
Thiel, 2004).
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Phages undergoing lytic cycles only are virulent. Lytic cycles consist
of several steps and show considerable variations according to the type of
phage. The general diagrammatic representation of the lytic cycle is as shown
under.
Phages encounter bacteria by chance and adsorb to specific receptors,
generally located on the cell wall, but also on flagella, pilli, capsules, or the
plasma membrane. The phage nucleic acid enters the host and the shell
remains outside. In phages with contractile tails, the cell wall is degraded by
phage enzymes located on the tail tip. The sheath then contracts and the tail
core is brought in contact with the plasma membrane.
It depends largely
on the physiological state of the host and varies between 20 min and 30–40
hrs. Phage nucleic acid is transcribed to mRNA using host and/or phage
RNA polymerases.
The assembly of new phages is called maturation.
Phage constituents assemble spontaneously or with the help of specific
enzymes (Ackermann and DuBow, 1987).
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The lysogenic cycle on the other hand comprises replication of phage,
nucleic acid together with the host genes for several generations with major
metabolic consequences for the cell. This is a latent mode of infection and it
occurs at a very low frequency. The phage gene in this state may
occasionally revert to lytic cycle.
Leading to release prophages particles.
This property is known as lysogens and phage that can develop both lytically
and lysogenically are said to be phages (Beneett and Howe, 1998).
2.3.2 Structure and Classification
Structure: All phages have single, linear double stranded DNA (dsDNA)
chromosomes stored in a protein coat shell called the capsid or head. The
capsid is built by protein molecules along icosahedrally symmetric arrays to
form the distinctive shape of phages. The tail extends from one corner of the
capsid and interacts with a single host cell. During infection, the distal end of
the tail adsorbs to the exterior of the host cell as phage DNA travels through
the tail into the cells (Ackermann and DuBow, 1989).
37
Antje et al. (1998) assigned the phages to different virus families,
species and strains based on morphology, DNA homology and host range.
The phenotypic diversity of the 22 bacteriophages was examined by electron
microscopy and phages were identified by following morphological criteria out
lined as per the International Committee of Taxonomy of Viruses.
Morphological studies of 22 phages detected by them revealed that all of the
phages
had
tails
and
thus
belong
to
the
order
Caudovirales.
The icosahedral heads of the phages had diameters between 50.2 and
99.3 nm. The phages could be assigned to three virus families. Eleven of the
phages belonged to the family Myoviridae, which contains phages that have
icosahedral heads and long contractile tails; seven phages were assigned to
the family Siphoviridae which contains phages that have icosahedral heads
and long flexible tails and four phages, which had icosahedral heads and
short tails, belonged to the family Podoviridae.
Phages belonging to 3 different families and their micrographs
illustrated by them are as under.
A. Myoviridae
B. Siphoviridae
38
C. Podoviridae
Electron microscopic images of the somatic coli phages (faecal coli
forms) isolated from environmental samples by Duran et al. (2002) were as
shown below. The structure (A) of bacteriophage with isometric head and
short tail indicates Myoviridae and another structure (B) of phage with long
straight flexible tail indicate the family Siphoviridae.
A. Myoviridae
B. Siphoviridae
Sandra et al. (2004) tested stool samples from pediatric diarrhoea
patients and environment water samples in Dhaka, Bangladesh and sewage
from Switzerland, for E. coli bacteriophages, yielded nearly exclusively
phages with a contractile tail belonging to family Myoviridae. The electron
microscopic structure and genomic DNA of the A, B and C are main three
selected phages based on their potential lytic activity on pathogenic E. coli
were as shown under. All three phages showed the typical morphology of
T4 like phages with 170 kb genome (D) upon pulsed field electrophoresis.
39
A
B
M
C
A B C
D
Verthe et al. (2004), examined electron microscopic images and
genomic DNA molecules of the phages while studying stability and activity of
an Enterobacter aerogenes–specific bacteriophage under simulated gastrointestinal conditions. Transmission electron microscopy revealed phage
particles having an isometric head (A) with a diameter of approximately 65 nm
and a short non-contractile tail. After restriction of phage DNA the size of the
40
major bands (B) were approximately 23,000 bp, 7000 bp and 4000 bp. These
morphological properties and the estimated genome size of at least 34 kb
correspond to the T7 like phages of the genus Podoviridae, family
Podoviridea and order Caudovirales.
A
B
Classification: The first global attempt to systematically classify viruses took
place at the International Congress of Microbiology held in Moscow in 1966.
This meeting established the International Committee of Taxonomy of Viruses
(ICTV), whose mission was to develop a universal taxonomic system for all
viruses infecting animals, plants, fungi, bacteria, and later, archaea.
Beginning with its first report in 1971, the International Committee of
Taxonomy of Viruses has met regularly to update virus definitions and
taxonomy guidelines, with the seventh and most recent report published in
2000 (Regenmortel, et al., 2000). Viruses are grouped together by shared
characteristics, with subgroups having smaller clusters of shared attributes.
For example, the tailed order of phage (Caudovirales) is broken down into
41
three families; phage with long, contractile tails (Myoviridae), phage with long,
noncontractile tails (Siphoviridae), and phage with short tails (Podoviridae).
The families are further broken down into genus and subgenus by criteria
such as genome configuration (linear, circular, super coiled), host range, and
genome size.
The inadequacy of the International Committee of Taxonomy of
Viruses classification is evident when one looks closely at the numbers.
Of the completed phage genomes currently deposited in GenBank (Benson,
et al., 2004), 40% (92 of 228) are unclassified beyond the level of family
according to International Committee of Taxonomy of Viruses conventions.
Furthermore, an additional 10% (23 of 228) are not even assigned an order
and are simply listed as “unclassified bacteriophage”.
It is generally agreed that future phage classifications must reflect
genomic data as a primary component. In bacteria, this is easily
accomplished by examining the conserved 16S ribosomal genes. However,
phage lack ribosomal DNA and there are no conserved gene or protein
sequences common to all phage on which to base a classification (Schuch
et al., 2002). Furthermore, any immediate attempt to reclassify phage based
exclusively on comparative genomics may be biased toward the lambdoid
phages or those involved in industrial fermentation, since these phages
dominate the current assemblage of sequenced genomes. Nonetheless, in
the fall of 2002, three separate phage research groups proposed alternative
classification schemes as outlined below.
42
An option proposed by Rohwer and Edwards is based on a “Phage
proteome tree”, which is constructed by grouping phage both relative to their
rear neighbours and in the context of all other phage (Rohwer and Edwards,
2002).
This method analyzed the entire predicted proteome for a given
phage and the results were then transformed into a distance matrix. A tree
was
constructed
based
on
relationships
between
phage
proteins.
The author list several anomalies in the International Committee of Taxonomy
of Viruses system that could be resolved by the proteome tree. Additionally,
the proteome tree moves, which International Committee of Taxonomy of
Viruses classifies as Tectiviridae due to the presence of a lipid membrane
below the capsid, to a subgenus of the family Podoviridae.
A second approach, put forth by a group at the Pittsburgh
Bacteriophage Institute, suggests that it may be impossible to have a strictly
hierarchical taxonomic system given the genetic mosaicism arising from
horizontal gene transfer among phage (Lawarence et al., 2002).
In this
paradigm, the top levels of taxonomy would still follow the hierarchical
Linnean approach, i.e., viruses would be divided into “domains” according to
genome type (double stranded DNA, single stranded DNA, Single–stranded
RNA, and double–Stranded RNA), with a further partition known as “divisions”
to separate defining characteristics such as tailed phage from filamentous
phage.
However, from this point on, three basic tenets would guide the
remainder of the classification.
First, members of a group should exhibit
similarity in one or more loosely defined cohension mechanisms. Second,
significant sequence data, preferably from whole genomes, should be
43
available for evolutionary assignment to a taxonomic cluster. Third, the
groups may be reticulate, i.e., phage may simultaneously belong to several
groups based on multiple and/or differing criteria from the first two tenets.
This web like design has an inherent flexibility that is not afforded by either
phenetic or genetic hierarchical approaches.
Finally, a third classification scheme, based on comparative genomics
of a structural gene module, has been proposed (Proux et al., 2002). Since it
is believed that the structural genes are the oldest and most conserved
module in dairy phage, dot plots of temperate lactococcal phage were used to
observe graded relatedness between DNA and protein sequences as well as
similarity in the organization of the structural genes in the absence of
sequence relatedness.
Whereas, comparative genomics of non-structural
genes tended to lump all of the lactococcal phages studied into one species,
comparative genomics of the structural genes delineated four phage species
and distinguished two genera based on head morphology. It may not be such
a bad idea that we agree to disagree (Daniel, 2004) with the exception of a
few remarkable outliners, some of which are noted above.
2.3.3 Sources and Distribution
Bacteriophages or “Phages” are viruses of prokaryotes including
eubacteria and archaebacteria. They were discovered and described twice,
first in 1915 by the British pathologist Frederick William Twort and then in
1917 by the Canadian bacteriologist Felix Hubert d’Herella working at the
Pasteur Institute of Paris. With about 3500 isolates of known morphology,
phages constitute the largest of all virus groups. Phages are tailed, cubic,
44
filamentous, or pleomorphic. Tailed phages are far more numerous than other
types, are enormously diversified, and must be very old in geological terms
(Richard Sharp, 2001).
Phages have been found in over 100 bacterial genera distributed all
over the bacterial world; in aerobes and anaerobes, actinomycetes,
archaebacteria, cyanobacteria and other phototrophs, endospore formers,
appendaged,
budding,
gliding,
mycoplasmas and chlamydias.
and
sheathed
bacteria,
spirochetes,
Phage like particles of the podovirus type
have even been found in endosymbionts of paramecia.
However, tailed
phages reported in cultures of green algae and filamentous fungi are probably
contaminants. Most phages have been found in a few bacterial groups:
enterobacteria
(over
650
phages),
bacilli,
pseudomonads, staphylococci, and streptococci.
clostridia,
lactococci,
This largely reflects the
availability and ease of cultivation of these bacteria and the amount of work
invested.
About half of phages have been found in cultures of lysogenic
bacteria. Tailed phages predominate everywhere except in mycoplasmas.
In archaebacteria, they have been found in the genus Halobacterium only and
not yet in methonotrophs and extreme thermophiles. Siphoviridae are
particularly
streptococci.
frequent
in
Myoviruses
enterobacteria,
actinomycetes,
and
pseudomonads,
coryneforms,
podoviruses
bacilli,
and
are
lactococcis
relatively
clostridia.
frequent
This
and
in
particular
distribution must have phylogeneic reasons.
Except for phages from extreme environments, phage species
generally seem to be distributed over the whole earth. This is suggested by
45
electron microscopic observations of rare and characteristical phage
morphotypes in different countries and global occurrence of certain
lactococcal phage species in dairy plants and of RNA coliphages in sewage.
Unfortunately, most data are from developed countries. Sizes of phage
populations are difficult to estimate because plaque assays and enrichment
and (most) concentration techniques depend on bacterial host; they therefore
only detect phages for specific bacteria and environmental conditions.
Consequently, phage titers vary considerably – for example, for coliphages
109/g in human feces, 107/ml in domestic sewage and 105/g of actinophages
in soil.
Several sources have been used by different researchers for the
isolation of bacteriophages and to understand their prevalence. Etwert (1980)
studied the distribution of phages in raw sewage and treated effluents. Bitton
(1987) also studied the distribution of phages raw domestic waste water.
Antije et al. (1998) and Yoon (1999) reported distribution or phages in sea
water and marine environment respectively.
Richard Sharp (2001) recorded the distribution of phages in sea water,
sewage and also soil. Keven et al. (2003) and Ghanaat et al. (2003) were
also recorded the prevalence of bacteriophages in different soil and sewage.
Bachrach et al. (2003) have isolated bacteriophages from human
saliva. Phages for Enterococcus faecalis were found in saliva samples. The
presence and stability of the E. faecalis bacteriophages in human saliva
suggests a possible role of these bacteriophages in a oral ecosystem and
phage therapy as a way to control oral bacteria might be considered.
46
Hitch et al. (2004) have isolated bacteriophages from the oral cavity. The
composition of the oral cavity does not appear to be heavily influenced by
interactions
between
bacteriophages
and
their
hosts
and
reported
bacteriophage for control of oral infections may need to be obtained from
other sources.
2.4 IN VITRO ACTIVITY OF BACTERIOPHAGES
Smith and Huggins (1982) while studying efficacy of phages against
pathogenic E. coli, isolated 15 phages from specimens of sewage, out of
which 9 were identified as specific phages based on the degree of the lytic
activity (diameter of plaques). The 9 anti E. coli phages were much more
virulent than the others based on in vitro lytic activity. 106–109 viable particles
of the phages were required to lyse broth of E. coli seeded with 3x108 viable
organisms. 50–120 resistant colonies were also observed within the zone
produced by 1 drop of undiluted preparations of few anti E. coli phages
spotted on a lawn of pathogenic E. coli, determining the difference in degree
of in vitro lytic activity of phages.
Prior observations of phage host systems in vitro have led to the
conclusion that, susceptible host cell population must reach a critical density
before phage replication can occur. Such a replication threshold or
“proliferation threshold” density would have broad implications for the
therapeutic use of phage. Kasman et al. (2002) were demonstrated
experimentally that, such replication threshold exists and support the
threshold in terms of a classical model for the kinetics of colloidal particle
interactions in solution.
47
An in vitro study was performed by Steven Hagens et al., (2004) to
develop genetically stable, efficient safe therapeutic phage for the infections
caused by P. aeruginosa. The use of technique minimized endotoxin release
and hence significantly reducing the side effects.
Flynn et al. (2004) have studied the exploitation of bacteriophage as
biocontrol agents to eliminate the pathogen E. coli. Two distinct lytic phages
isolated against a human strain of E. coli and a cocktail of phages were
evaluated for their ability to lyse the bacterium in vivo and in vitro. However,
bacteriophage – insensitive mutants emerged following the challenge and
commonly reverted to phage sensitivity within 50 generations.
Tanji et al. (2004) screened from various sources, 26 phages infected
with E. coli. Among them 9 caused visible lysis of E. coli cells in Luria Bertani
liquid medium.
However, prolonged incubation of E. coli cells and phage
allowed the emergence of phage resistant cells.
A bacteriophage showing high in vitro lytic activity against a clinically
important strain of Enterobacter aeruginosa was isolated from hospital
sewage by Verthi et al. (2004). The stability and lytic activity of phage against
the clinical isolate under simulated gastro–intestinal in vitro conditions was
also evaluated.
Sandra et al. (2004) studied in vitro bacteriolytic activities of E. coli
phages to understand implications of phage therapy. 40 phages specific to
pathogenic strain of E. coli were isolated from stool samples of pediatric
diarrhea patients and environmental water samples in Dhaka, Bangladesh
and sewage from Switzerland. 4 selected phages based on prominent plaque
48
features were rescreened for their lytic potential on the E. coli by a tube lysis
test. This test is more labour intensive than the spot test but, offers a more
rigorous assessment of bacterial lysis in vitro activities of the test phages.
2.5 FACTORS INFLUENCING PHAGE ACTIVITY
Eric et al. (1996) studied effects of sunlight on the viability and
structure of bacteriophage in marine aquatic systems. Destruction of virus
particles is concluded to be a process separate from loss of infectivity. It is
also concluded that strong sunlight affects the viability of bacteriophages in
surface waters, with the result that direct counts of virus like particles over the
estimate of bacteriophage capable of both infection and replication. However,
in deeper waters, where solar radiation is not a significant factor, direct counts
should more accurately estimate numbers of viable bacteriophages.
Yoon et al (1999) were suggest that, a novel bacteriophage,
designated as VPP97 which infects the of Vibrio para haemolytics (hallophilic,
Gram-negative bacteria) isolated most commonly from marine environments.
In this studies characterized phages were almost totally inactivated at 70oC
and at pH below 5 or over 10, and the phage treatment appears effective to
the infection by V. parahaemolytics.
Feng et al. (2003) studied effects of pH and temperature in the survival
of coli phages. MS2 phage survived better in acidic conditions than in an
alkaline environment in contrast Q  phage had a better survival rate in
alkaline conditions; than in an acidic environment. The inactivation rates of
both coli phages were lowest within the pH range 6-8 and the temperature
range 5-350C. The inactivation rates of both coli phages increased when the
49
pH was decreased to below 6 or increased to above 8. The inactivation rates
of both coli phages increased with increasing temperature. Substances or
conditions that denature proteins or react chemically with proteins or nucleic
acids will in activate phages. The inactivation of MS2 and Q  observed in
their study could be attributed to reactive radicals and levels of heat stability.
Temperature has a major effect on the effectiveness and/or the rate of kill or a
given microorganism because it controls the rate of chemical reactions. Thus,
as temperature increases, the rate of kill induced by a chemical will also
increase. In addition, pH can affect the ionization of chemicals.
At
extreme pH values, the high concentrations of hydrogen ion and hydroxyl ion
present in water are considered to be far greater than the concentration of
free reactive radicals and therefore dominate viral inactivation mechanism.
Verthi et al. (2004) studied on effect of pH, bile salts and pancreatin on
bacteriophage in simulated gastro-intestinal in vitro conditions. After
1 hour incubation at 34oC at different pH levels, there were no significant
differences between the initial phage titer of 6.2  0.3x105 pfu/ml and the
phage concentrations at pH 9, 7, 6 and 4. However, at pH2, the concentration
of bacteriophage dropped below the detection limit (1.0x101 pfu/ml)
immediately after the addition of phage.
2.6 IN VIVO EFFICACY OF BACTERIOPHAGE
Smith
and
Huggins
(1982)
studied
successful
treatment
of
experimental E. coli infections in mice using phages and their general
superiority over antibiotics.
The minimum lethal dose (LD50) of pathogen
E. coli was 3x107/ml and phage was 3x108/ml.
50
The phage administered
intramuscularly
3-5
days
before
challenge
with
a
potentially
intramuscularly induced infection of MW was protective.
lethal
However they
observed variations in protective effect of phages propagated on different
pathogenic strains of E. coli. A single intramuscular dose of anti E. coli phage
was more effective than multiple intramuscular doses of tetracycline,
ampicillin, chloramphenicol, or trimethoprim plus sulfphfurozole in curing mice
of a critically induced infection of pathogen E. coli. The few phage resistant
mutants of E. coli were also observed during the study. According to them
3x103 to 3x104 phage particles were sufficient to cure mice given a potentially
lethal dose of pathogenic E. coli.
Soothill (1992) examined bacteriophage for P. aeruginosa in
experimental infections of mice to investigate their potential for the treatment
of infection of man.
A Pseudomonas phage protected mice against
LD50–(8x107 cfu/ml) of a virulent strain of a P. aeruginosa with a LD50 of
1.2x107 particles. This studies support the view that bacteriophages could be
useful in the treatment of human infections caused by antibiotic resistant
strains of bacteria.
Gowri Sankar et al. (1998) evaluated phage therapy to treat
experimental infections in mice due to a clinical isolate of P. aeruginosa,
which
was
isolated
from
patients
admitted
into
a
rural
hospital.
Bacteriophage specific for this pathogen was isolated from a domestic
sewage. Test animals (mice) were injected with the bacterial pathogen
(0.1 ml suspension with 1.75x104 cells/ml) through the intraperitoneal route.
Their phage patterns were also administered 0.5-1.0 ml suspension with
51
(5.5x104 pfu/ml). Subsequently through the same route after a time lap of
6 hours. Animals protected with phage ingestion exhibited no sign of illness
when compared to the unprotected ones. This protection was attributed to
the marked reduction (from 6.3x108/g to 4.0x105/g of viscera of mice) of the
pathogen load in the tissues of the animals in response to the presence and
persistence of phage.
Karen et al. (2002) studied phage therapy of local and systemic
disease caused by Vibrio vulnificus in mice. In this study, they examined the
potential use of bacteriophages as therapeutic agents against V. vulnificus in
an iron-dextran-treated mouse model of V. vulnificus infection. Mice were
injected subcutaneously with 10 times the lethal dose V. vulnificus and
injected intravenously, either simultaneously or at various times after infection,
with phages. Treatment of mice with phages could prevent death; systemic
disease, as measured by cfu/g of liver and body temperature; and local
disease, as measured by cfu/g of lesion material and histopathologic analysis
were carried out. Samples of the infected blood, liver and peritoneal lavage
fluid were sampled at various intervals, after injection of the phage, and
pfu/ml or g were enumerated. Data was expressed in means and standard
deviations.
Biswajit et al. (2002) reported that bacteriophage therapy rescues mice
bacteremic from a clinical isolate of vancomycin resistant Enterococcus
faecium.
Ghanaat et al. (2003) have investigated the potential of phages for the
treatment of P. aeruginosa infection in mice. A strain of P. aeruginosa was
52
used
which
was
resistant
to
many
or
routinely
used
antibiotics.
Bacteriophage was originally isolated from sewage by standard methods
using mentioned strain of P. aeruginosa as host (Slopek et al., 1987).
Pathogen free mice were chosen at 3rd wks. of age. First approximate lethal
dose 50% of this strain of P. aeruginosa were surveyed (around 105 cfu). In
their experiment, a much higher dose (107 cfu) could kill 100% of non-treated
mice. Two groups of 10 mice (case and control) were inoculated
intraperitoneally with 107 cfu of P. aeruginosa. In the case group, 109 pfu of
phage particles was inoculated intraperitoneally cavity, at the meantime and
every 12 hr upto 4 doses.
After inoculation of mice with 107 cfu of P. aeruginosa, they became ill
and eventually, collapsed within 24 hr of inoculation. When both
P. aeruginosa and phage (107 cfu of bacteria and 109 pfu of phage) were
administered simultaneously followed by 4 other doses of 109 pfu of phage
every 12 hr, no mortality was observed at all. The numbers of dead mice in
the control and treated groups were 10 and 0, respectively. They concluded
that
in
mice,
which
inoculated
with
P.
aeruginosa,
bacteriophage
administration showed significant protection against mortality.
Studying the kinetics of bacterium and phage multiplication in these
mice showed that in the absence of phage the P. aeruginosa multiplied in
mice almost as fast as in broth culture. When phage was given to these mice
intraperitoneally, the phage multiplied rapidly on the bacteria and prevented
massive multiplication, resulting in a decline in bacterial numbers.
53
Naturally occurring, antibiotics resistance bacteria were used to
measure the growth of phage in vivo by Richard et al. (2004). E. coli RRU
and the pathogenic K-1-capsualted E. coli ATCC 23503 were used as host for
phages T4 and K1-5. 107 log-phase bacteria were employed. Newly weaned
Rattus rattus rats were used as experimental animals oral doses were
administered observations were made in the digestive system. For phage
and bacterial samples, rats were killed, dissected, the guts sectioned and the
contents removed by squeezing.
Four T4–like coli phages with broad host ranges for diarrhoea
associated E. coli serotypes were isolated by Sandra et al. (2004) from stool
specimens of pediatric diarrhoea patients and from environmental water
samples. All four phages showed a highly efficient gastrointestinal passage
in adult mice when added to drinking water. Viable phages were recorded
from the faeces in a dose dependent way. The minimal oral dose for
consistent faecal recovery was as low as 103 pfu of phage/ml of drinking
water. In conventional mice, the orally applied phage removed restricted to
the gut lumen and as expected for a non-invasive phage, no histopathological
changes of gut mucosa were detected in the phage-exposed animals. E. coli
strains introduced into the intestines of conventional mice and traced as
ampicillin – resistant colonies were efficiently lysed in vivo by phage added to
the drinking water.
54