July 21, 2009 : An interesting Piece of News

From:

ClinicalTrials.gov

Experimental Phage Therapy of Bacterial Infections

This study is currently recruiting participants.

Development of a phage cocktail (SSP TM) effective against highly virulent strains of Shigella and Salmonella spp

From:

US.ARMY and here and Food safety


Shigella

A Prospective, Randomized, Double-Blind Controlled Study of WPP-201 for the Safety and Efficacy of Treatment of Venous Leg Ulcers. Phase I

An interesting piece of news:

From:
Journal of Wound Care, Vol. 18, Iss. 6 , 01 Jun 2009, pp 237 - 243

D.D. Rhoads, R.D. Wolcott, M.A. Kuskowski, B.M. Wolcott, L.S. Ward, A. Sulakvelidze


Objective: This phase 1 trial set out to examine the safety of a bacteriophage based preparation for difficult to treat wounds.

Method: The intention to treat sample comprised 42 patients with chronic venous leg ulcers (VLUs); 39 patients completed the trial. The ulcers were treated for 12 weeks with either a saline control or bacteriophages targeted against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Follow-up continued until
week 24.

Results: No adverse events were attributed to the study product. No significant difference (p>0.05) was determined between the test and control groups for frequency of adverse events, rate of healing, or frequency of healing.

Conclusion: This study found no safety concerns with the bacteriophage treatment. Efficacy of the preparation will need to be evaluated in a phase II efficacy study.

Declaration of interest: One of the authors (AS) holds an equity interest in Intralytix. The other authors do not have any interest in commercial activities.




Study Phase


These phases are defined by the Food and Drug Administration (FDA) in the Code of Federal Regulations.


Most clinical trials are designated as phase I, II, III, or IV, based on the type of questions that study is seeking to answer:

Phase I

In Phase I clinical trials, researchers test a new drug or treatment in a small group of people (20-80) for the first time to evaluate its safety, determine a safe dosage range, and identify side effects.

Phase II

In Phase II clinical trials, the study drug or treatment is given to a larger group of people (100-300) to see if it is effective and to further evaluate its safety.

Phase III

In Phase III clinical trials, the study drug or treatment is given to large groups of people (1,000-3,000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the drug or treatment to be used safely.

Phase IV

In Phase IV clinical trials, post marketing studies delineate additional information including the drug's risks, benefits, and optimal use.



From Clinical Trials.gov.:

"A Prospective, Randomized, Double-Blind Controlled Study of WPP-201 for the Safety and Efficacy of Treatment of Venous Leg Ulcers"

Sponsored by: Southwest Regional Wound Care Center
ClinicalTrials.gov- Identifier: NCT00663091


Phase I : completed (April 14, 2009 )



Drug: Bacteriophage

WPP-201 is a pH neutral, polyvalent phage preparation, which contains 8 bacteriophages ("component bacteriophages" or "component phages") lytic for P. aeruginosa, S. aureus, and (Table 1). The E. coli cocktail contains a concentration of approximately 1 x 10^9 PFU/ml of each of the component monophages. The phage component of WPP-201 is roughly estimated to be 0.5 ppm by weight and the remainder is phosphate-buffered saline containing < 1,000 ppm total organic carbon from the growth medium and biomass. All phages contained in the preparation have been originally isolated from the environment, and they have not been genetically manipulated in any way (i.e., the preparation is 100% natural). WPP-201 contains no preservatives and antioxidants.

Does Phage go in or does Phage not go in through the Blood?

Two conflicting Viewpoints



From The Bacteriophages.org

The distribution of phage pfu's in mice following various routes of administration

This graph was adapted from data from a 1973 experiment in which germ free mice were inoculated with a single dose of 2X10^12 pfu Lambda phage. In these experiments oral administration of phage resulted in the detection of a systemic level of phage tissue titers that were 7 to 8 orders of magnitude lower than that achieved by systemic administration of phage.

Graphic representation of data from the 1943 infectious disease model in which mice were inoculated by intracerebral injection of the bacteria Shigella dysenteriae (at an LD50 level) were compared with uninfected control mice.

Graph

All of the mice in this experiment were injected with 10^9 pfu of phage i.p. which was administered at the same time as the bacterial inoculation. The bacteriophage level in the blood of the uninfected animals was compatible with the dilution of the phage concentration in the total fluid volume of the mouse and the lower levels in the brain reflect the relatively smaller blood content in the brain. However, in the infected animals the phage particles are observed to increase at the site of the infection, the brain, while the blood levels of phage appear to be a 'reflection of the events occurring in the brain.

Graphical representation of data presented by Smith and Huggins. (In this set of experiments all of the animals received an intracerebral inoculation of 5X10^2 cfu of an E. coli K1).

The animals treated with phage were injected with 3X10^8 pfu of phage intramuscularly (into the gastrocnemius muscle) at the same time as the bacterial inoculation. These graphs were derived from the data published in tables 9 and 10 in their paper.

Endotoxin Removal

Endotoxin Activity in a Coliphage Preparation


PREPARATION OF ENDOTOXIN-FREE BACTERIOPHAGES
JANUSZ BORATYŃSKI, DANUTA SYPER, BEATA WEBERDĄBROWSKA,
MARZANNA ŁUSIAK-SZELACHOWSKA, GRYZELDA POŹNIAK and ANDRZEJ GÓRSKI.


Methods of Endotoxin Removal from
Biological Preparations:
Pérola O. Magalhães, André M. Lopes, Priscila G.
Mazzola, Carlota Rangel-Yagui, Thereza C. V. Penna,Adalberto Pessoa Jr.


EndoTrap®: highly specific endotoxin removal without incubation

Endotoxin Removal Products



From: www.plosone.org

“Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials”

Economics of Antibiotic Resistance

From Expert Review of Anti-Infective Therapy

Economics of Antibiotic Resistance

by Oguz Resat Sipahi

Published: 09/30/2008


"The importance of phage therapy and associated research may increase proportionately with the incidence of pandrug-resistant microorganisms. Although it may be considered a kind of individualized therapy and may need complex technical equipment, the cost of phage therapy was found to be less than vancomycin, linezolid, teicoplanin and quinopristin/dalfopristin in the treatment of staphylococcal infections.[138]"

The Temporariness's Principle

Bacterial host cells are not defenceless against phage attack. The heavy burden put on the susceptible bacteria may select cell variants that are refractory to bacteriophage infection (bacteriophage insensitive mutants, BIMs). This is usually accomplished by loss, modification,or masking of the bacteriophage receptors located at the cell wall.

A number of strategies that may be used to overcome or limit resistance development have been indicated in the literature, including the prevention of the recycling of the bacteriophages in the reservoir of the pathogen by alternating use of different bacteriophages (either in a cocktail of several bacteriophages, or in consecutive treatments).


Bacteriophages generally exhibit a narrow host range, which is usually restricted to one genus of bacteria but more frequently restricted to either a limited number of species within a genus or to a limited number of bacterial strains within a species .
The best virulent bacteriophages for Phage Terapy applications are those with the broadest possible host range. These are termed polyvalent bacteriophages or WHR (wide host range) bacteriophages as they are usually active against many species within a bacterial genus.

Phage Therapy

a)when we do not know if the bacterial infection is caused by one bacterium or by two or three bacteria and when we have very little time.

Phage Therapy administration:

Bacteriophage composition (cocktail of 4 or 8 or 10 different Phages; cocktail of 6 or 9 or 12 different Phages)


b)when we have time and when we know the bacterial cause of infection.

If the infection is caused by bacteria:

Phage Therapy administration:

Bacteriophage composition (cocktail of 4 or 8 or 10 different Phages; cocktail of 6 or 9 or 12 different Phages)


If the infection is caused by one bacterium:

Phage Therapy administration:

Bacteriophage composition (cocktail of 2 different Phages; cocktail of 3 different Phages; cocktail of 4 different Phages)

The Temporariness's Principle

The Effectiveness of Bacteriophage compositions is conditioned by the Temporariness's Principle:

"The Effectiveness of Bacteriophage compositions ready to use, when they are used in Phage Therapy, must be tested frequently. Both Host range and Virulence of any Bacteriophage composition must be unchanged when it is compared to respective original composition".

Each Bacteriophage composition is designed to match regional strains of bacteria for different infections in different parts of the body. We must test the sensitivity of phages to dominant strains of bacteria every 5-6 months. The Phage cocktail is updated by adding new phages or removing old ones to attack newly emerging strains.

It is important to understand that the precise properties exhibited by one bacteriophage cannot be assumed to be identical for other bacteriophages. Each bacteriophage will have its own characteristic properties including host range, burst size, and ability to maintain its physical integrity in different environments.

A Focal Point: Exclusion of Temperate Bacteriophages from the Host Strains

From:

"Bacteria, Phages and Septicemia

Aušra Gaidelyté 1, Martti Vaara 2, Dennis H.Bamford 1
1 Department of Biological and Environmental Sciences, Institute of Biotechnology, University of Helsinki, Helsinki, Finland, 2 Department of Clinical Microbiology, Helsinki University Hospital, Helsinki, Finland

We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease.
Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis causing bacterial clones play a role in clonal selection during bacterial invasion.”

Methods for Phage induction

1-For UV induction, bacteria were grown to 200 Klett units and collected by centrifugation for 10 min at 6,000 rpm using a Sorvall SS-34 rotor at 4°C. Bacteria were suspended in the same volume of M9 broth and transferred to a glass Petri dish.
The bacterial suspension was irradiated for 42 sec at A254 followed by dark storage on ice for 1 h. Cells were collected by centrifugation for
5 min at 13,000 rpm using a Heraeus Biofuge at 22°C. Bacteria were suspended in 3 volumes of LB and the number of plaques was determined after additional two h incubation at 37°C.

2a-For MitC (mitomycin C) induction experiments, cells were grown to 200 Klett units and induced with MitC at a final concentration of 5 mg/ml. Cells were incubated for 15 min at 37°C and the growth medium was then replaced with fresh LB. Plaques were determined after additional two h incubation at 37°C.

or

2b-To confirm the absence of temperate bacteriophages,originating from the bacterial hosts a standard technique for bacteriophage induction using the DNA-damaging antimicrobial agent mitomycin C was carried out, as described by Miller.
Bacterial cultures were aliquoted in 1 ml volumes in sterile eppendorf tubes, covered with aluminium foil thus protecting the bacteria from photoreactivation of drug-induced DNA damage. Mitomycin C (Sigma-Aldrich) was added to final concentrations of 1 or 5 mg/ml . A control tube without mitomycin C was added to evaluate the presence of ‘nondrug induced’ bacteriophages. The tubes were incubated for 3 h at 37°C. Subsequently, twenty ml of chloroform was added to the control tubes to lyse the bacteria. The lysates were centrifuged in order to separate the intact bacterial cells from the supernatant. The final titre of bacteriophages in the supernatant was determined using the double agar overlay method.

3-To test if antibiotics could induce phage production, the same procedure was used as in the previous paragraph with the following
modifications. Three different antibiotic concentrations were tested depending on the antibiotic used and bacterial strain employed.
For E. coli strains, 1, 10, and 30 mg/ml final concentrations of tobramycin (tomycin, Orion Pharma) and 0.03, 0.3, and 3 mg/ml of ciprofloxacin (Bayer) were used. For S. aureus strains, 1, 10, 20 mg/ml final concentrations of tobramycin and 1, 10, 30 mg/ml of ciprofloxacin were used.

Viable counts of cell suspensions were determined to evaluate the antibacterial activity of UV, MitC or antibiotic treatment.

Bacterial Virulence properties altered by Phages

From:

Bacteriophage Control of Bacterial Virulence
Patrick L. Wagner and Matthew K. Waldor

Phage Therapy Preparations

-Natural Phages or Natural mutant Phages

-Phages that are modified by genetic engineering

-Virolysins (phage-encoded lytic enzymes also called lysins or endolysins)


From:
Recent Patents on Biotechnology 2009, 3, 000-000
"Production and Application of Bacteriophage and Bacteriophage-Encoded Lysins"
Noémie Manuelle Dorval Courchesne, Albert Parisien, Christopher Q. Lan
Department of Chemical and Biological Engineering, The University of Ottawa, Ottawa, ON, Canada K1N 6N5

".... it was reported that sub-lethal concentrations of certain antibiotics could substantially stimulate the host bacterial cell's production of virulent phages. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic E.coli strain's production of the phage MFP by more than 7 fold. This phenomenon, which is designated as Phage Antibiotic Synergy (PAS), was observed in diverse hostphage systems. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. The PAS phenomenon was found to be directly related to the formation of filamentous (elongated) cells under the stress of sub-lethal antibiotics. It was hypothesized that there has been an evolutionary selection for phages that can more efficiently cannibalize host cells that are unable to further divide. It was further suggested that if this is true, then the optimal conditions for phage multiplication may not be exponential cell growth as is widely believed, but rather a terminal burst of phage production in a stressed cell population that would soon die anyway. This observation provided the foundation for two recent patents exploiting the PAS phenomenon for enhanced phage production.

Applications of phages and virolysins include treatment and prevention of bacterial infections, detection of pathogens in foods and other samples, and decontamination of foods and medical devices.
Phages can also be utilized for a diversity of other applications such as in targeted drug delivery and in preventing biofilm formation in industrial processes".

Phage Therapy in Burn Wound Patients : an excellent Work

From: www.plosone.org

“Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials”

This scientific work points out all steps(*) for producing a Bacteriophage Cocktail used for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients.

-*Titration of bacteriophage suspensions using the agar overlay method

-*Initial isolation, separation and purification of P. aeruginosa and S. aureus lytic bacteriophages

-*Production of bacteriophage stocks

-*Selection of therapeutic bacteriophages

-*Composition and endotoxin purification of the bacteriophage cocktail

-*Final quality control

-*Pyrogenicity

-*Sterility

-Transmission electron microscopy

-*Exclusion of temperate bacteriophages from the host strains

-*Genome sequencing and proteomic analysis

-*Cytotoxicity towards keratinocytes

-*Stability at 4°C

-*Stability after spraying

-*Batch record file

-*Clinical application

Mutation of a Phage Receptor and its Phenotypical Delay

From:

“-Second, and more limiting, the development of resistance. Even when the appropriate bacteriophage is applied to the appropriate host, the host bacterium has the ability to mutate its receptor and enlist other defenses to evade the phage. This problem can ben circumvented by using multiple phages, identifying new phages or trying to mutate the original treating phage to recognize the changed host bacterium.”


This point may cause misunderstandings about the efficacy of the Phage therapy:

the host bacterium has the ability to mutate its Phage receptor (example:the receptor for one single phage) but the genetic mutation for changing this receptor present on the bacterial cell wall (about 200 copies for one phage) requires a long time and several replications.

This effect is an example of Phenotypical Delay.

Mutation Rate or Mutation Frequency

A mutation rate is an estimation of the probability of a mutation occurring per cell division and corresponds to the probability of a mutation occurring in the lifetime of a bacterial cell.
A mutation frequency is simply the proportion of mutant bacteria present in a culture.

These terms are often used interchangeably, causing confusion.

The relationship between mutation frequency and the rate at which mutations occur is uncertain.

If a mutation arises early in the culture period, then a large number of mutant progeny occur and this would be represented by a high frequency.

This phenomenon is known as a “jackpot culture” and was first described in 1943 by Luria and Delbrück during their seminal set of experiments investigating the mutation of Escherichia coli from bacteriophage T1 sensitivity to resistance.

Fluctuation Test of Luria and Delbrück

Luria and Delbrück demonstrated that bacteriophage-resistant mutant colonies arise from a sensitive culture of E. coli if bacteriophage T1 is present in excess. Resistant colonies appeared from sensitive cultures, i.e., in which there was clearing,within 12 to 16 h. These bacteria were resistant to bacteriophage T1 but sensitive to other viruses capable of causing lysis in that strain of E. coli.
Luria and Delbrück showed that reversion to sensitivity was a rare event and that, in a growing culture, the proportion of resistant bacteria increased with time. They argued that if the presence of the phage was needed to trigger the change to resistance, then the distribution of mutant colonies should demonstrate a Poisson distribution.
The high variance in the numbers of mutants in the culture,however, led Luria and Delbrück to conclude that resistant mutants were present in the culture before bacteriophage exposure and that the bacteriophage resistance mutation arose independently. The Luria-Delbrück distribution is different from the Poisson distribution in that its variance is greater than 1.
Luria and Delbrück assumed that for a bacterium there was a small fixed chance that a resistance-conferring mutation could occur per unit of time if the bacteria are “in an identical state.” The number of mutated cells in a culture depends on how early the mutation occurred during the growth of the bacterial population. If mutation occurs early in the culture, the number of mutated cells will be higher than if it occurs later.

By the Fluctuation Test is possible to calculate the Mutation Rates and to determine the rate of mutation to Resistance to phage.

Phage Therapy and Human Wound Infections

Bacteriophages

Bacteriophages (phages) are viruses that only infect their host bacteria.

Bacteriophage has specialized absorption structures, which bind specific receptors on the host bacteria. This specific binding gives most phages a very narrow host range, which is usually just a small subpopulation (strain) of a particular species of bacteria.

Once the absorption takes place, the phage is able to inject its genetic material into the host bacteria. A transition then takes place by which the phage derived genetic material takes over the host’s metabolic systems and reproduces phage components. The different parts of the phage are then assembled into whole functional phages.

There are a few studies looking at the ability of bacteriophage to infect host bacteria residing in a biofilm phenotype. Several studies in controlled laboratory settings have demonstrated phage can absorb, replicate and reinfect their host bacteria within a biofilm structure.

Specific phage products such as polysaccharide depolymerase have been identified, and have been observed to degrade the host bacterium’s EPS material (extracellular polymeric substances). However, the EPS material produced by a particular bacterium changes based on substrates and interactions with other bacterium in the biofilm. So it is unclear in a clinical setting how effective phage will be against biofilm.

Two points need to be made in defense of the use of bacteriophage in treating biofilm-based diseases.

-First, there are an estimated 10^31 bacteriophages worldwide. This makes bacteriophages a highly successful “life form” whose only food source is their host bacteria. Bacteria in natural environments overwhelmingly choose to live in an organized biofilm. Therefore, bacteriophages must have some natural success infecting biofilms.

-Second, there seems to be a significant clinical success in treating biofilm-based diseases such as sinusitis, osteomyelitis, wounds and other infections with bacteriophages.

In an over 60-year history in using bacteriophages clinically, there have been multiple clinical studies and case reports suggesting the efficacy of bacteriophage in biofilm infections. Although there is no controlled randomized study to date, clinical evidence suggests that bacteriophage is indeed effective against biofilms.

The use of bacteriophage in the management of chronic wounds has several advantages. Bacteriophage is specific for its particular host, will amplify itself indefinitely as long as the host bacteria is available and requires lysis (death) of the bacterium. Also, bacteriophage is extremely safe with no significant adverse events reported in human use.

Two disadvantages of bacteriophage have limited its use in therapy.

-First, bacteriophage’s narrow host spectrum requires a number of different individual phages against a particular species (example: P. aeruginosa). And even with phage cocktails, that is, polyvalent phage mixtures, there are still gaps in the treatment spectrum. For example, in P. aeruginosa a standard phage cocktail, (polyvalent phage) will cover only about 80 percent of pseudomonas strains. This is comparable, however, to many antibiotics.

-Second, and more limiting, is the development of resistance. Even when the appropriate bacteriophage is applied to the appropriate host, the host bacterium has the ability to mutate its receptor and enlist other defenses to evade the phage. This problem can ben circumvented by using multiple phages, identifying new phages or trying to mutate the original treating phage to recognize the changed host bacterium.

These two problems, along with the inherent advantage of the biofilm structure preventing absorption of the phage, makes phage therapy in human disease a little more challenging, but not impossible.

In phage therapy for chronic wounds several strategies have been pursued.

-First, debridement of the wound causes a significant disruption of the physical barriers to absorption and improves the kill rate of the phage.

-Second, combining phage therapy with antibiotics, such as macrolides (which disrupt the EPS material) or other antibiotics that challenge the environmental edge may make the biofilm more susceptible to phage penetration.
Also, multiple applications, or even continuous application, of phage may overcome some of the problems with absorption and, therefore, infection of the biofilm.

Phage products such as depolymerase, holins, and lysins are being developed and studied for their possible use in infectious diseases.

Also, more targeted research is being done on broader host range phages, developing cocktails with better phage synergies and more virulent phages.