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The first detailed images of an elusive drug that targets the outer wall of bacteria may provide scientists with enough new information to aid design of novel antibiotics. The drugs are much needed to treat deadly infections initiated by Staphylococcus aureus and other bacterial pathogens.

The research team, led by Natalie Strynadka, a Howard Hughes Medical Institute (HHMI) international research scholar at the University of British Columbia in Vancouver, Canada, published its findings in the March 9, 2007, issue of the journal Science.

“This enzyme is an awesome target for antibiotics. We have a totally new understanding of how the enzyme works and how a very good animal antibiotic inhibits the enzyme”, Dr Strynadka said.

Penicillin and many newer antibiotics work by blocking a piece of the machinery bacteria use to construct their durable outer walls. Without these tough, protective coatings, bacteria die. The enzymatic machinery (known as PBP2) studied by Strynadka’s group has two main parts: One end assembles long sugar fibers; the other end stitches them together with bits of protein to form a sturdy inter-locking mesh shell.

Strynadka’s team has provided a long-awaited look at the portion of the enzyme used in the first step of the biochemical pathway that initiates assembly of the sugar coating. The second step is targeted by penicillin and has been well studied.

Although scientists have spent many years identifying bacterial components whose structural features might have weaknesses that can be exploited by antibiotics, progress in turning up bona fide drug targets has been slow. The cell wall enzymes in particular have tantalized scientists, Strynadka said. “The cell wall has all the hallmarks of a great drug target,” she explained. “It is essential to the survival of all bacteria. The enzymes that create the cell wall are unique to bacteria. And it is accessible; you don’t have to get the
antibiotics into the cell.”

In their structural studies, the researchers focused on Staphylococcus aureus, a notorious human pathogen. An epidemic strain of the bacteria known as methicillin-resistant Staphylococcus aureus is resistant to several common antibiotics, including penicillin and amoxicillin, and is a great cause for concern among hospital infectious disease staff.

Postdoctoral fellow Andrew Lovering, who is first author on the paper, hopes the group’s three-dimensional pictures of the sugar-building enzyme from S. aureus will accelerate the search for an effective weapon against the infamous super bug.

The images produced by Strynadka’s team show the enzyme frozen in place by a powerful antibiotic called moenomycin. Moenomycin has been used for decades in animal feed to promote livestock growth. Bacteria have shown very little evidence of resistance to this antibiotic so far, and scientists think related compounds may be promising candidates for use in humans.

“This enzyme is an awesome target for antibiotics,” said Strynadka. “We have a totally new understanding of how the enzyme works and how a very good animal antibiotic inhibits the enzyme.” Although moenomycin is poorly absorbed by the human body, the new understanding of exactly how it
interferes with bacterial enzyme function should help scientists design modified versions that are more suitable for use in people.

Understanding the structure of this enzyme should also speed up screening and design of new antibiotics, which are in constant demand as microbes continually evolve new ways to evade the drugs that researchers design to thwart them.

The time it takes for bacteria to develop resistance to new antibiotics has been as short as one year for penicillin V and as long as 30 years for vancomycin.

Researchers attempting to solve the structure of this enzyme have struggled to recreate its cellular environment in the laboratory. But after much tinkering with different combinations of detergent, ions, and chemical additives, Strynadka’s team was able to crystallize the enzyme so that it would diffract x-rays into a pattern that would ultimately reveal its natural structure. They then were able to repeat the feat to reveal the crystal structure of the enzyme combined with the animal antibiotic.

Their findings help reveal how the enzyme prepares to assemble the bacteria’s sugar-coating by plucking sugars from a fat-sugar package known as lipid II. The antibiotic, which is another kind of sugar-lipid, probably mimics the lipid II molecule by tucking into a fold in the enzyme and taking up the space needed to bind to lipid II, the researchers believe. “We would like to see the enzyme in a complex with its natural substrates as well as with inhibitors,” Lovering said.

In the meantime, scientists now have the details of its shape and key contact points between enzyme and
antibiotic. The enzyme structure is the first ever solved of a member of a family of enzymes that remove sugars from lipids and attach them to other sugars. This process is used in a wide range of biochemical reactions, including allergic responses and cell signaling in cancer.

Researchers from the University of Sheffield have received joint funding from the Engineering and Physical Science Research Council (EPSRC) and the Ministry of Defence (MoD) to develop an innovative sensor to detect bacteria. The new method will use a polymer which will give a fluorescent signal when it encounters bacteria, allowing scientists to easily identify infected wounds much earlier than using conventional methodologies.

The new technology will be of immediate benefit to healthcare industries in general, as well as those involved in detecting infection in battlefield conditions and bacterial contamination, whether accidental or deliberate.

Currently identifying bacterial infection takes several days and requires swabbing and culturing of bacterial swabs as well as the use of specialist bacteriology laboratory facilities.

By combining polymers, which change shape when they encounter bacteria, and developing a light signal through fluorescence non radiative energy transfer (NRET), the researchers will be able to detect early stages of bacterial contamination.

Being developed by a multi-disciplinary team of researchers from the University’s Departments of Chemistry, Engineering Materials and the Dental School, the sensor will have widespread applications beyond the initial project.

Dr Steve Rimmer from the University’s Department of Chemistry, said: “The project is a great example of progress that can be achieved at the life sciences/physical sciences interface and we hope the project will deliver technology of real importance.”

The multi-disciplinary team will be led by Dr Steve Rimmer of the Department of Chemistry and consists of Dr Linda Swanson (Chemistry) Professor Sheila MacNeil (Engineering Materials) and Dr Ian Douglas (Clinical Dentistry).

The project received £670,000 funding jointly from the Engineering and Physical Science Research Council and the Defence Science and Technology Laboratory – an agency of the Ministry of Defence over three years and started in December 2006.

Over the past twenty years, the rapid emergence and increased prevalence of opportunistic Gram-negative bacilli demonstrating resistance to the β-lactam class of antibiotics has become a major health care crisis.

The production of β-lactamases, the innate capabilities of these organisms to genetically adapt structural and regulatory genes and the ease with which resistance genes are transferred via plasmids, transposons and integrons between different species, have broadened the ability of Gram-negative bacteria to inactivate the β-lactam antibiotics. This diminishes the clinical utility of these key anti-microbial agents making them resistant.

Extended spectrum β-lactamases (ESβLs) hydrolyse the penicillins, first-, second- and third-generation cephalosporins, especially cefotaxime, ceftriaxone, ceftazidime and cefpodoxime, and the oxyimino-monobactam, aztreonam.

ESβLs are inhibited by β-lactamase inhibitors, such as clavulanic acid, and are susceptible to the carbapenems (imipenem, meropenem and ertapenem) and the cephamycins (cefoxitin and cefotetan), though there have been a number of reports stating that ESβL-producing organisms can become resistant to the cephamycins due to the loss of an outer membrane porin protein (Martinéz-Martinéz et al 1996).

Since their discovery following the clinical introduction of the third-generation oxyimino-cephalosporins in 1981, there are now approximately 160 Temoneira (TEM), 100 sulfhydryl-variable (SHV), 64 cefotaxime-hydrolysing (CTX-M) and 102 oxacillinase (OXA) variant enzymes, along with a number of minor ESβL variants (Jacoby and Bush 2007).

Extensive laboratory and clinical experience exists regarding the detection and treatment of ESβL-producing Gram-negative bacilli. This suggest that the knowledge of their existence via means of antibiotic selective pressure, adaption and dissemination, may have an impact on therapeutic choices and the health and well-being of patients via targeted pragmatic antimicrobial selection and infection control practices.

It is unclear; however, if ESβL-producing organisms are being accurately detected 100% of the time. Furthermore, with the recent emergence of metallo β-lactamase-producing Gram-negative bacilli, it is also unclear whether the same mandate exists for the accurate detection, treatment and control of metallo β-lactamases. Metallo β-lactamases (MβLs) are a therapeutic disaster.

These enzymes hydrolyse all β-lactam antibiotics (except the monobactams), including the “drugs of last resort” the carbapenems (imipenem and meropenem), thus requiring the use of alternative, potentially more toxic classes of antibiotics to circumvent the hydrolytic actions of these β-lactamases.

Metallo β-lactamases, which are found in organisms such as Pseudomonas aeruginosa, Acinetobacter specie and members of the Enterobactericeae group such as salmonella and especially Escherichia coli and Klebsiella pneumoniae. They all utilise metal ions (usually zinc) to coordinate water molecules that serve as nucleophiles and hydrolyse the amide bond of the β-lactam ring, rendering the β-lactam antibiotic inactive.

These enzymes are divided into four genetically mobile variants: the older imipenem-hydrolysing (IMP) and Verona integron-encoded metallo β-lactamase (VIM) enzymes; and the more recently described Sao Paolo metallo β-lactamase (SPM) and GIM types (Poirel et al 2004).

Gram-negative bacteria that produce extended-spectrum and metallo β-lactamases are being discovered and isolated at a significant rate worldwide, while the development of new synthetic and natural antimicrobial agents to combat and elude the hydrolytic actions of these β-lactamases has significantly decreased in recent years (Valenzuela et al 2004).

Clinicians prescribing antibiotics need to know, understand and appreciate the short and long term outcomes of the inappropriate use of antibiotics for their patients, which, if not controlled and decreased, will inevitably reduce or eliminate the therapeutic options available in the future.

References

Franklin, C., Liolios, L., Peleg, A.Y. (2006). Phenotypic detection of carbapenem-susceptible metallo β-lactamase-producing Gram-negative bacilli in the clinical laboratory. Journal of Clinical Microbiology, 44: 3139-3144.

Martinéz-Martinéz, L., Hernández-Allés, S., Albertí, S., Tomás, J., Benedi, V., Jacoby,G.A. (1996). In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy, 40, pp. 342-348.

Poirel, L., Heritier, C., Spicq, C., Nordmann, P. (2004). In vivo acquisition of high-level resistance to imipenem in Escherichia coli. Journal of Clinical Microbiology, 42 (8), pp. 3831-3833.

Valenzuela, J., Thomas, L., Iredell, J. for Australian Society of Microbiology (ASM). (2004). Beta-lactam resistance in Gram-negative bacteria. Antimicrobial Susceptibility Testing: Methods and Practices with an Australian Perspective, 5, pp. 127-157.

Alaska Food Diagnostics claims that its Fastrak Salmonella test, can produce results six hours earlier than other comparable test methods including the TECRA and BAX.

By reducing the time of the salmonella tests, processors can save hundreds of thousands of dollars as any delays in their delivery time impacts on their efficiency.

The group of Alaskan scientists, working at UK government’s Defense Science and Technology Laboratory in Northern Ireland, have exploited adenylate kinase (AK) phage technology to develop the highly sensitive and specific fastrAK rapid assay system, the company claims.

The Fastrak Salmonella is based on established bioluminescence technology fastrAK™ which uses immunomagnetic beads to attract and isolate target pathogens and then AK-Phage® technology employs bacteriophage that infect the target organism. As the bacteriophage multiplies the bacteria rupture releasing Adenylate Kinase (AK). The AK converts ADP to ATP which reacts with luciferase to produce measurable light.

The only hurdle is passing the rigorous AOAC and AFNOR validations that proves is effectiveness and compete with other cost effective test method.

Pradip Patel, head of microbiology research and development at Alaska said the speed of the assay is a real breakthrough, along with its accuracy.

‘”We are confident it will make a significant impact in the drive for improved food safety standards” he said.

Sample pre-enrichment for 16 hours can be initiated throughout the day, ready for testing the next morning, the manufacturer said.

After just two hours, products with known quality control results can be shipped in time to meet ready-to-eat poultry standards, Alaska said.

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Biophage Pharma Inc has report its first financial result for 2006 – 2007. The company is a high-potential, revenue-driven biotechnology company focused on the development of an integrated approach for the prevention and control of bacterial infections within the food industry.

Overview of operations
Biophage reached an important milestone in its Biosensors Division in the first quarter of 2007 in the development of its compact PDS96 (R) Biosensor. The Corporation is now conducting extensive in-house performance testing and validation of this alpha prototype. Biophage also furthered the development of its four new biosensors: The BacTrapping(R) system, the micro-fluidics system, the “FastBac” biosensor and the e.sensor. In this context, Biophage signed an important collaborative agreement aimed at combining a2sp’s Magic Tag(R) immobilization technology with Biophage’s biosensor platform. Magic Tag(R) uses linkers, which are activated by daylight, for the immobilization of
biomolecules (including phages) onto different surfaces such as magnetic beads, biosensors and micro-array surfaces. Biophage and a2sp also jointly filed a patent application on February 16, 2007, relating to “methods for immobilizing viruses (phages) using photo-reactive linkers”.

In the Therapeutics Division, Biophage concluded initial sales of its LISTEX(TM) product to an important cheese producer in the U.S. Securing this
sale marked the beginning of a business relationship with our first client who intends to develop and use phage therapy as a biological solution to control potential Listeria monocytogenes (Listeria) contamination in cheese. On December 4, 2006, Biophage signed an MOU (memorandum of understanding) with EBI Food Safety (La Hague, Netherlands) for the sale and distribution of LISTEX(TM) in North America. LISTEX(TM) is the first bacteriophage product to receive FDA GRAS (Generally Recognized as Safe) recognition for the control of Listeria contamination in cheese.

In ImmunotoxLabs, Biophage hired Dr. Michel Heyne as director of its Beryllium Reference Lab to meet the increasing demand for Beryllium and MELISA(R) testing. With his vast experience in laboratory testing, this eminent hematologist will help expedite the accreditation process of
Biophage’s Beryllium laboratory by the Quebec National Institute of Public Health (INSPQ).

Financial Results
Contract revenues for the three months ended February 28, 2007 amounted to $107,551 compared with $195,944 in the same three month period in fiscal 2006. The decrease in substantially attributable to the completion of significant projects with important clients, although partially offset by an increase in revenues generated from Beryllium testing. Other income for the first quarter in 2007 reached $824 compared to $601 in the same period in fiscal 2006.

Research and development costs for the three months ended February 28, 2007 (before tax credits) amounted to $135,006, representing a $61,303, or 83% increase over the $73,703 recorded in the same interim period in the preceding fiscal year. The increase is substantially attributable to the hiring of additional staff affected to R&D, including a director for the Corporation’s Biosensors Division, commensurate with Biophage’s overall accelerated efforts in developing the phage therapy segment. Research and development tax credits for the first quarter amounted to $35,000, which compares to $20,000 for the three month period ended February 28, 2006, representing 26% and 27% of
related costs, respectively.

Costs of contracts for the three months ended February 28, 2007 amounted to $117,410, relatively unchanged from the $118,359 incurred during the same period in the preceding fiscal year. The slight decrease in the costs of contracts results from lower subcontracting and laboratory supply costs, which was almost entirely offset by an increase in salaries from the hiring of additional staff between the interim periods.

Biophage’s net loss for the three month period ended February 28, 2007 amounted to $261,287 ($0.01 per share) compared to a net loss of $157,821 ($0.00 per share) for the corresponding three month period in the preceding fiscal year.

Liquidity and Financial Resources
As at February 28, 2007 Biophage had cash and cash equivalents of $426,733 compared to $214,344 at November 30, 2006. The increase in cash and cash equivalents from November 30, 2006 levels is substantially attributable to the private placements completed during the interim period, although partially offset by cash used in operating activities (after changes in non-cash working capital items).

During December 2006 and February 2007, the Corporation issued 4,045,458 units through private placement. Each unit is made up of one common share and one common share purchase warrant, whereby each common share purchase warrant is exercisable for a period of two years at an exercise price of $0.17 per common share. The 4,045,458 shares were issued for a total cash consideration of $525,910. More detailed information regarding the foregoing can be found in the interim unaudited consolidated financial statements and related management
discussion and analysis which have been filed today on SEDAR at www.sedar.com.

Granting of Stock Options
On April 27, 2007, the Corporation’s Board of Directors granted stock options to purchase an aggregate 1,114,000 common shares of the Corporation at an exercise price of $0.12 per share to certain directors, employees and consultants of the Corporation, all of which vest immediately other than 150,000 options that will vest on the first anniversary of the grant and 150,000 options that will vest on the second anniversary of the grant. The grant of such stock options is made in accordance with the stock option plan of the Corporation. The granted options will expire on April 27, 2012.

About Biophage Pharma Inc.
Biophage Pharma is a high potential, revenue-driven Canadian biotechnology company focused on the development of innovative phage-based
products and technologies for the detection, prevention and control of bacterial infections. Founded in 1995, Biophage operates three divisions:

(1) The Biosensors division for the development and commercialization of Biosensors, more particularly a portable PDS96(R) Biosensor which is now in the pre-commercialization stage; (2) The phage therapy division for the prevention and control of bacterial contaminations in the medical, veterinary and environment fields; (3) The Immunotox Labs division, which provides services in Immunogenicity and Immunotoxicity, Beryllium sensitivity testing and MELISA(R) testing for the detection of sensitization to more than 200 different allergens including metals, penicillin, gluten and pollens.

Source: www.biophagepharma.net www.immunotoxlabs.com

A multiple antibiotic resistant strain of pseudomonas aeruginosa known as (Methicillin Resistant Pseudomonas Aeruginosa (MRPA) has forced the cancellation of some elective cardio thoracic and neurological surgery cases in the Royal North Shore (Australia).

Because of this outbreak, the intensive care unit remained closed this week until the bacteria is eliminated from the area.

The neurological surgery ward (6C) of the Intensive Care Unit within the hospital was closed prior to Easter and patients were moved to another section of intensive care while cleaning and sanitation of the area took place.

Environmental swabs are currently being tested for the suspect bacterium and the ward is expected to be re-opened this week once the area is confirmed free of this strain of bacterium.

MRPA was isolated and identified on 10 patients during the late March and this prompted the management of the hospital to take aggressive measures including isolating infected patients before closing the affected ward and basically bombing the area with super grade disinfectant.

Pseudomonas aeruginosa is commonly found in soil and water; however there are a few strains that have started to build up resistant against front line antibiotics. Once these strains multiply and become the dominant type within the environment, we have a real problem especially if patients using life-saving antibiotics are located in the same area.

According to a spokesperson within the hospital, they say that the hospital usually has about 30 to 40 cases of MRPA a year, mostly in intensive care wards.

Patients with confirmed MRPA were being cared for in isolation with treatment for their primary illness continuing without interruption. Their families were permitted to visit but had to follow infection control procedures such as washing hand before and leaving the hospital grounds.

Did you know that money can be a source of micro-organisms. Although low, the cross-transmission of micro-organisms to foods can occur through food handlers.

A group of researchers in Ballarat , Australia screen 400 coins and 350 notes for the presence of bacteria. The money was sourced locations where staffs are likely to handle money. This included small food outlets such as corner shops, cafes and bakeries.

From the findings and as expected, the most common bacterium isolated was Staphylococcus aureus; a micro-organism commonly present on the skin and nasal passages of a third of the human population.

Pathogens were also isolated with E.coli dominating a high proportion of the coins. Only 2 coins had salmonella.

Although the majority of the money contained micro-organisms, the levels were very to cause direct infections.

The biggest risk is the cross contamination of low levels of micro-organisms to foods that support the growth. This is the reason why food handlers must wash their hands properly and regularly or glove wearers to changes gloves frequently.

It looks like the giant US company 3M has just taken over the British Biomedical firm “Acolyte Biomedica” which makes microbial detection technology.

According to sources at 3M, the acquisition will help its expansion into the market for infection prevention diagnostics.

One of the firm’s major products is a rapid test system that helps hospitals screen for Methicillin Resistant Staphylococcus Aureus (MRSA), a type of bacteria that is resistant to many antibiotics and occurs most frequently in hospital patients who have weakened immune systems.

The system lets physicians screen for MRSA or other types of resistant bacteria and get confirmed negatives in hours instead of days. This acquisition adds to the purchase of Biotrace in 2006 has a similar type of technology. Interestingly Biotrace also recently took over the popular Australian Biotechnology company, TECRA. TECRA is one of the world leaders in rapid tests kits for pathogen testing the food industry.

Here’s what they write in the press release:

“Early detection of dangerous microbes is becoming more important as multiple resistant bacteria strains become more prevalent. Acolyte Biomedica helps hospitals control high-risk infections through improved screening and targeted treatment of methicillin-resistant Staphylococcus aureus (MRSA), a type of bacteria that is resistant to certain antibiotics and occurs most frequently in hospital patients who have weakened immune systems.

Complementary acquisitions such as this support both 3M’s core business and growth strategy to expand into adjacent markets. 3M’s long-standing infection prevention platform offers innovative solutions to help reduce the risk of healthcare-associated infections. 3M’s infection prevention portfolio includes diagnostic testing, sterilization assurance, skin preparation, sterile field and surface, wound management and environmental cleaning.”

According to the Sydney Morning Herald, the source of the Legionella outbreak has been linked to the Royal Automobile Club (RAC) building in Macquarie Street. The link is still to be confirmed.

It is interesting what they said a few days ago when the possible source could have been eliminated as the infection occurred around the New Year period. Since then the regular cleaning would have eliminated the source. It looks like there going to be a surge of Legionlla testing this year.

Anyhow here’s what they write:

“Seven cases of the potentially fatal disease have been reported in NSW this month, including six patients who were in the Circular Quay area on New Year’s Eve.

Outbreaks are usually associated with readings above 1,000.”

According to Lisa Finneran from the DAILY PRESS (NEWPORT NEWS, VA.), the rise of Drug-Resistant Staphylococcus Infections is growing worst each day. She writes:

“Kathleen Jaeger thought she had survived the worst condition of her life: a breast cancer diagnosis, chemotherapy and a double mastectomy.

Although the community-contracted bacterium is resistant to many drugs, there are some older drugs in the penicillin family that can successfully combat the bug.”

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