July 21, 2009 : An interesting Piece of News

From:

ClinicalTrials.gov

Experimental Phage Therapy of Bacterial Infections

This study is currently recruiting participants.

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.

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.

What are the main Features for selecting Bacteriophages for Phage Therapy?

We must consider:

Phage Host range

(a mathematical criterion is required for selecting an useful phage among a lot of phages)

A selected Phage must have a Wide Host Range.

A bacteriophage with a wide host range is a phage capable of killing the highest number of different isolated cultures belonging to a given bacterial species ( example: Staphylococcus aureus from various sources).

For this reason it is indispensable to apply a mathematical criterion for selecting a phage among a lot of phages capable of effectively killing bacteria of a targeted bacterial species from various sources.

Virulence

A bacteriophage is selected as “Virulent “ when this phage is capable of effectively killing bacteria from various sources compared to capacity of the non selected phages.

For selecting a phage, by this property, we use the criterion of the lower concentration capable of killing bacteria compared to concentrations of the non selected phages.

Example:

two Phages with the same Host range

Phage A is effective in killing bacteria at 10^7 dilution
Phage B is effective in killing bacteria at 10^9 dilution

Phage B is the selected phage.

Mutant Phages

Selection of a Mutant Phage by Mutagenization:

a) phages must be always lytic phages

b) mutant phages, if compared to wild-type phage ( non mutaginezed phage or Parental Phage), may have both host range and virulence increased.

How many Types of Phage we must use for a Phage Therapy Treatment?

Phages are "bacterium specific" and it is therefore necessary in many cases to take a swab from the patient and culture it prior to treatment.
Occasionally, isolation of therapeutic phages can typically require a few months to complete, but clinics generally keep supplies of phage cocktails for the most common bacterial strains in a geographical area.

The host specificity of phage therapy may make it necessary for clinics to make different cocktails for treatment of the same infection or disease because the bacterial components of such diseases may differ from region to region or even person to person.

In addition, due to the specificity of individual phages, for a high chance of success, a mixture of phages is often applied. This means that 'banks' containing many different phages are needed to be kept and regularly updated with new phages, which makes regulatory testing for safety harder and more expensive.

Phages in practice are applied orally, topically on infected wounds or spread onto surfaces, or used during surgical procedures. Injection is rarely used, avoiding any risks of trace chemical contaminants that may be present from the bacteria amplification stage,and recognizing that the immune system naturally fights against viruses introduced into the bloodstream or lymphatic system.

Phages can usually be freeze dried and turned into pills without materially impacting efficacy.In pill form temperature stability up to 55°C, and shelf lives of 14 months have been shown.

Oral administration works better when an antacid is included, as this increases the number of phages surviving passage through the stomach.

Topical administration often involves application to gauzes that are laid on the area to be treated.

Other forms of administration can include application in liquid form. These vials are usually best kept refrigerated.
The lytic bacteriophages available for phage therapy are best kept refrigerated but discarded if the pale yellow clear liquid goes cloudy.

Phage therapy is generally considered safe. As with antibiotic therapy and other methods of countering bacterial infections, endotoxins are released by the bacteria as they are destroyed within the patient (Herxheimer reaction). This can cause symptoms of fever.

Care has to be taken in manufacture that the phage medium is free of bacterial fragments and endotoxins from the production process.

Monomicrobic Infection caused by one Bacterium

For example: a Mix of 3 lytic Phages for Bacterium A

Phage 1
Phage 2
Phage 3


Phage Receptor Types and Receptors Number (?) on the cell wall, example for 3 Phages
(3 different Receptors):Receptor 1(X?), Receptor 2(Y?), Receptor 3(Z?)


Rate of Mutation to Resistance to Phages:
Phage 1(10^-7), Phage 2(10^-7), Phage 3(10^-7)

Rate of Mutation to Resistance to all lytic Phages for Bacterium A:
(10^-7)*(10^-7)*(10^-7)=10^-21

When, for example, a priori we suspect an Infection caused by 3 different Bacteria:
Bacterium A, Bacterium B, Bacterium C

For example: a Mix of 6 (or 9) lytic Phages (2 or 3 Phages for each bacterium):

For Bacterium A

Phage A1
Phage A2

For Bacterium B

Phage B1
Phage B2

For Bacterium C

Phage C1
Phage C2


Phage Receptor Types and Receptors Number (?) on the cell wall, example for 6 Phages (2 different Receptors for each bacterium ):

Bacterium A

Receptor A1(X1?)
Receptor A2(X2?)

Bacterium B

Receptor B1(Y1?)
Receptor B2(Y2?)

Bacterium C

Receptor C1(Z1?)
Receptor C2(Z2?)



Rate of Mutation to Resistance to lytic Phages for each bacterium:

Bacterium A

Phage A1 (10^-7)
Phage A2 (10^-7)

Bacterium B

Phage B1 (10^-7)
Phage B2 (10^-7)

Bacterium C

Phage C1 (10^-7)
Phage C2 (10^-7)


Rate of Mutation to Resistance to lytic Phages:

Bacterium A =(10^-7)*(10^-7)= 10^-14
Bacterium B =(10^-7)*(10^-7)= 10^-14
Bacterium C =(10^-7)*(10^-7)= 10^-14

The discovery of a natural phenomenon: PAS (Phage-Antibiotic Synergy). Implications for Phage Therapy.

Phage-Antibiotic Synergy:Implications for Phage Therapy


La « synergie phages-antibiotiques » : Un enjeu pour la phagothérapie

Acinetobacter baumannii (MDR), first part

Colonies are 1 to 2 mm, nonpigmented, domed, and mucoid, with smooth to pitted surfaces.





Acinetobacter baumannii: An Emerging Multidrug-resistant Threat


Wikipedia:Acinetobacter baumannii







Acinetobacter spp.


Acinetobacter

Example of trend in Phage Therapy research

OBJECTIVE: Develop an antibiotic solution or substance consisting of least three different lytic bacteriophages that kill Acinetobacter baumannii for eventual use in human wound infections.

Development of Bacteriophage Therapy for Treatment of A. baumannii Infected Wounds

Some Phage Therapy works

M. Abhilash, A. Vidya & T. Jagadevi : Bacteriophage Therapy: A War Against Antibiotic Resistant Bacteria. The Internet Journal of Alternative Medicine. 2009 Volume 7 Number 1



Production and Application of Bacteriophage and Bacteriophage-Encoded Lysins



Bacteriophages: Beyond Antibiotics



Bacteriophage Therapy



The Next Phage


Viruses could kill superbugs that antibiotics can't

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".

Sources and Bacteriophages isolation

Bacteriophages can be relatively easily isolated from various sources.

The best source of a given phage is material where its specific host is abundant.

Bacteriophages active towards pathogenic microflora are usually acquired from the material obtained from a given patient (urine, faeces, pus, etc.). The other alternative which further gained wide spread is the isolation of virulent phage isolates from sewage waters (normally from the clinic).Thus, for phages infecting the human gut flora, municipal sewage is an excellent source.

Before starting isolation of a Bacteriophage, its bacterial host needs to be isolated and grown in pure culture. During phage isolation, it is generally best to use host bacteria in the exponential growth phase, although some phages also appear able to propagate in bacteria in the stationary growth phase.

The growth conditions (medium, oxygen level, temperature, incubation time, etc.) should be chosen according to the preference of the host bacterium.

Phages can be isolated from various aqueous (e.g., water) and nonaqueous (e.g., soil) sources. If phages are to be isolated from nonaqueous samples, the samples first should be suspended in a medium suitable for the growth of the targeted bacterial species.

To remove solids, indigenous bacteria, and
other organisms, the samples can be “clarified.” This step can be accomplished by centrifugation (1500g, 20 min), which will remove most of the bulk solids, after which the supernatant can be processed for phage isolation.

Filtering the suspended samples through a 0.45 mm or 0.2 mm membrane filter (preferably a tangential flow filter, to avoid rapid clogging) is an alternative approach, but this step may still need to be preceded by a centrifugation step or by pre-filtering the suspensions through larger-pore filters. Sometimes samples can be processed directly for phage isolation without clarification.

Enrichment of the Bacteriophages in the source material

Enrichment of the phages in the source material often facilitates phage isolation.

The ratio between source material and added (concentrated) growth medium should be varied to accommodate differences in abundance of phages and the nature of the source material, as should the addition of host bacteria. If one wants to enrich for particularly virulent, broad-spectrum phages that initially constitute a very small fraction of the phages in the sample, the protocol should utilize a larger volume of
source material, several different host bacteria, and several cycles of selection.
If the volume of the sample is large compared to the final culture volume, however, some of the sample’s components may interfere with bacterial growth.

That problem can often be circumvented if the volume of source material is ≤1%–10% of the culture’s final volume.

If the sample constitutes more than 10% of the final volume, one should add enough 10X culture medium to have the final enrichment culture contain 1X culture medium.

There are a lot of protocols, for example:

5oo ml of municipal sewage is mixed with 100 ml of 10X Bacterial growth Broth and with 1X Bacterial culture medium. This sample is incubated overnight at 37°C and after the Phage Titre value
(= Plaque Number*Reciprocal of Diluition*Reciprocal of Volume in ml ) is calculated by Titration of bacteriophage suspensions using the agar overlay method.


Chloroform is often used to break open infected cells, since only cells whose peptidoglycan layer has been weakened by
phageencoded lysozyme or endolysin will be lysed by chloroform. However, it should be omitted from all procedures where phage with lipid-containing envelopes are expected or sought.

Once isolated, phages can be resuspended in phage diluent (TSG or SM), or growth medium.