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The effort to find preserved samples of the 1918 influenza virus has been a pursuit of both historical and medical importance.

The influenza pandemic in 1918 was the most devastating single disease outbreak in modern history, and examining the virus that caused it may help prepare for, and possibly prevent, future pandemics. When the complete sequence of the 1918 virus was published in 2005, it represented a watershed event for influenza researchers worldwide.

An article in the journal Antiviral Therapy, scientists at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, narrate the story of how scientists discovered samples of the 1918 strain in fixed autopsy tissues and in the body of a woman buried in the Alaskan permafrost.

The article places this discovery in the context of decades of research into the cause of pandemic influenza, and the authors detail the strange convergence of events that allowed them to recover and sequence the virus in the first place. Its genetic material is so fragile that it should not have survived for days, let alone decades.

In a mass grave in a remote Inuit village near the town of Brevig Mission, a large Inuit woman lay buried under more than six feet of ice and dirt for more than 75 years. The permafrost plus the woman’s ample fat stores kept the virus in her lungs so well preserved that when a team of scientists exhumed her body in the late 1990s, they could recover enough viral RNA to sequence the 1918 strain in its whole entirety. This remarkable good fortune enabled these scientists to open a window onto a past pandemic. It could also help mankind gain a foothold for preventing a future one.

Did you know that bacteriophages which are commonly known as “phages” are naturally occurring viruses that infect and kill bacteria with very high specificity.

They do this by attaching to the surface of the bacterium, replicating inside, and eventually destroying their host. Most importantly, bacteriophages are specific for only one type of bacteria so the normal flora are left intact. Because replication is so rapid (one phage can produce 2 billion offspring in 2 hours) bacteria have little opportunity to develop resistance. In addition, once the target bacteria have been destroyed, the phages are no longer capable of reproduction, and subsequently disappear through natural processes, leaving no harmful residues. This environmentally friendly characteristic of phages is a major advantage when compared to conventional antibiotic use, where toxic residues have led to many problems. As such they represent a formidable, yet underutilised weapon in our constant war against bacterial infections.

Bacteriophage Therapy is the practical application of these very powerful lytic viruses to a bacterial infection, whether in animals, fish or even plants. The concept may appear novel but the fact is that it has been used for over 85 years in Eastern European countries like Georgia and Poland where it became part of the standard health care to treat burns, wound infections and gastrointestinal disorders.

Bacteriophages were discovered independently by two scientists between 1915 and 1917, more than 20 years prior to the isolation of Penicillin.

In 1915, Frederick Twort reported an “ultracosmic virus that somehow killed bacteria in solution”. Two years later Felix d’Hérelle a French-Canadian biologist identified and coined the name bacteriophage, meaning “bacteria eater”. Highly excited by the efficiency of the viruses against Shigella bacteria, d’Hérelle continued his studies on phages and was the first person to realise the potential of bacteriophages as therapeutic agents. In 1919 for the first time in history, he treated a 12 year old boy with severe dysentery; within 5 days of treatment the boy was completely cured.

After d’Herelle’s first successful use of phage therapy, other scientists around the world became interested in the new phenomenon and its potential as a therapeutic method.

Europe and the United States began to produce their own phages on a large scale for the medical treatment of cholera, typhoid fevers and bubonic plague. In 1932 in India alone, 191,000 vials of bacteriophages were used for the treatment of cholera. However, at the time, very little was known about phage biology and the very high specificity required for a successful treatment was poorly understood. Many famous scientists, including Bordet, disagreed violently with d’Herelle that the phenomenon was caused by a virus and argued that it was an enzyme or an active component present in the solution which caused the observed effect. Others thought that the solution simply stimulated the immune system, facilitating the healing process. Due to the resulting confusion doctors often used mismatched phages with a corresponding lack of success. Companies manufacturing the phages compounded the problem by making exaggerated claims and supplying poor quality products. It was hardly surprising that negative reports began to appear in the literature questioning the effectiveness of phage therapy.

By the late 1940’s, mired in controversy and with the widespread availability of penicillin and other broad spectrum antibiotics, phage therapy fell into decline and eventually vanished from the Western scientific radar, except as a bacteriology typing tool and as a platform for molecular biology. After all, the successful use of phage therapy depended heavily on the correct identification of a particular bacterial pathogen, plus the skills required for production. Why bother when the “magic bullet”, penicillin, came as a white powder, had broad spectrum of activity and was cheap and highly effective?

Fortunately, scientists in the former Soviet Union and various Eastern European countries, including Georgia and Poland persevered with their studies of phages. In Georgia, The Eliava Institute has been producing phages for the treatment of patients since 1930 and has recently attracted the attention of scientists anxious to benefit from their vast experience in the treatment of bacterial infections. However, despite the fact that phage therapy has been widely used and refined for over eighty years, it is still considered by Western science to be an experimental field because much of the development preceded the modern, more stringent regulatory standards of western pharmaceutical products.

A number of research institutions and companies around the world have taken up to the challenge of re-introducing bacteriophage therapy to our markets for the treatment of human, animal and agricultural bacterial infections. Initial studies have confirmed many of the original scientific observations. As a result, clinical trials have been set up in England, Germany and the United States to study the efficacy of phages. In the agricultural sector, the first phage based product for the use on tomatoes and pepper crops has received a commercial registration from the U.S Environmental Protection Agency (EPA). The FDA has also granted approval for a new core product of 6 lytic phages as a food additive in meat and poultry products to prevent infection with Listeria monocytogenes, a bacterial pathogen that affects more than 2500 people annually, especially pregnant women, newborns and immune-depressed individuals. In Wroclaw, the Institute of Immunology and Experimental Therapy (IITD) has been granted authorization to treat people with phages in cases where all else had failed.

The encouraging developments show that Phage Therapy has the potential to be a useful alternative antimicrobial therapy. However, much work still needs to be done to optimise the treatment protocols and to provide solid evidence on the safety of the treatment to the highest standards. Nonetheless, with the inevitable rise in antibiotic resistance and the diminishing pipeline of new antibiotics, phage therapy may prove to be a valuable and timely weapon in the fight against bacterial infections.

About the authors
Dr. Anthony Smithyman completed a PhD on Bacteriology and Immunology at Glasgow University in 1978. For the past 20 years he has managed Cellabs Pty, a Sydney-based diagnostics company, which specialises in Tropical and Infectious diseases.

Sandra Morales is a Microbiologist working for Special Phage Services Pty Ltd, Australia’s first phage-therapy company. She is also a PhD student in the University of Technology of Sydney and is currently undertaking an investigation of the potential use of bacteriophages in the treatment of antibiotic resistant infections. The project includes the screening of hundreds of Australian isolates against a broad collection of bacteriophages and studying their efficiency and potential for viable products.

References
EARSS Management Team, members of the Advisory Board, and national representatives of EARSS. (2006) EARSS Annual report 2005, On-going surveillance of S. pneumoniae, S. aureus, E. faecalis, E. faecium, E. coli, K. pneumoniae and P. aeruginosa. Bilthoven, Netherlands.

Häusler, T. (2006) Viruses vs Superbugs A solution to the antibiotic crisis? Houndmills, Basingstoke, Hampshire RG21 6XS and 175 Fifth avenue, Ney York, N.Y 10010: Macmillan.

Hayden, M.K. et al (2005) Development of Daptomycin Resistance In Vivo in Methicillin-Resistant Staphylococcus aureus. Clin Microbiol.; 43(10): 5285–5287

Kutter, E. & Sulakvelidze, A. (2005) Bacteriophages Biology and Applications. United States of America. CRC.

Noskin, G MD. et al. (2005) The Burden of Staphylococcus infections on Hospitals in the United States: An analysis of the 2000 and 2001 Nationwide Impatient Samples Database. Arch Inter Med, 165(1):1756-1761

Stone, R. (2002) Stalin’s forgotten cure The Forgotten Cure. Science, 298(5594): 728-31.

Sulakvelidze, A., Z, A. and G, M. (2001) Bacteriophage Therapy: Minireview. Antimicrob Agents Chemother, 45, 649-659.

Sulakvelidze, A. (2005) “Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections.” Drug Discovery Today 10(12): 807-809.

This biofilm-producing terrorist is the bane of all industrial microbiologists. Industry can be humming along quite happily and then up pops Listeria and its panic stations. The micro response team rushes to the site armed with gauzes, swabs, sampling and HACCP (Hazard Analysis and Control of Critical Points) plans to do combat.

As we all know the genus Listeria is a gram positive rod, psychrotrophic, and displays a peculiar tumbling motility caused by a low number of peritrichous flagella which beat in a clockwise motion due to a defective CheY gene (Dons et al, 2004). This organism is ubiquitous and is found primarily in soil (Sutherland et al, 2003). The only species that is truly a human opportunistic infector is Listeria monocytogenes, public enemy number one. Its sibling Listeria ivanovii is attempting to cause confusion in the ranks of those over-worked industrial microbiologists. L. ivanovii has shown similar pathogenicity as seen by L. monocytogenes, in mice and other animals, but is rarely seen in humans (FDA/CFSAM, 2003). Are these two species protected or masked by Listeria innocula the harmless one? With the perceived threat of Listeriosis, the government bodies are debating the move towards zero tolerance for the genus. The federal government food body FSANZ standard only states that L. monocytogenes absence is required in ready-to-eat products and the FAO/WHO risk assessment concluded that levels of L. monocytogenes <100 cells per gram has the same risk as zero cells per gram (FAO/WHO, 2001). To complicate matters, Dussarget (2004) stated that of the 13 known serovars of L. monocytogenes, only 1/2a, 1/2b and 4b are responsible for 98% of reported human Listeriosis cases. The serovar 4b is associated with the majority of food borne outbreaks and sporadic cases. This single genus has been responsible for more product recalls and media hype than any other micro-organism. We all have heard of Conroy’s and the two deaths from ham in Adelaide in the last few months. Industries that produce ready-to-eat products all have great concern for this ubiquitous terrorist.

Industry has spent millions on the combat, control and the eradication of this organism. As with all terrorist organizations, the sleeper cells are very hard to find and the fact that Listeria produce a fatty acid biofilm on solid surfaces makes it very difficult to treat with standard chloride based surface sanitizers. This biofilm aids the survival of Listeria due to its lipid composition which is hydrophobic and thus prevents the entrance of water-based sanitizers; it also acts as a food reserve and selects for the survival of other symbiotic organisms that aid in the survival and proliferation of Listeria (Sutherland et al, 2003) (Somers & Wong, 2004). The destruction of one biofilm may lead to the establishment of others from that original source and to product contamination. Biofilms are living entities and thus, when critical mass is achieved, cells detach and contaminate the product. This is known to the industrial microbiologist as ‘spitting’. There is reported resistance developing in the standard chemicals used in the eradication of biofilms (Chavant, 2004). The only effective way to clean down contaminated areas is by high-pressure (area needs to be sealed) acid washes as well as physical scrubbing followed by contact sanitization (quats, chlorine, acid and peroxide sanitizer) – the chemical equivalent of hunting down terrorist cells with thermonuclear warheads. Listeria has also displayed an ability to survive and thrive in some of the most extreme environments found in industry such as saturated brine. Listeria has been associated with many of our most loved and highly consumed foods. These include: ice cream, soft cheeses, smoked salmon, pate, fermented meats, cooked further processed chicken meats and fresh leaf produce (Sutherland et al, 2003). This cowardly bacterium attack the elderly, infirmed and the defenseless fetus with relatively low infective doses, 2 to 3 log less than is required to infect healthy adults (CFSAN, 2003). To complicate matters further, this organism presents to the treating clinician as flu -like systems and initial diagnosis may be difficult.

The total number of victims recorded in Australia is 3 cases in 1,000,000 and is steadily decreasing as the industrial microbiologist is slowly eradicating all known niches. The consumers demand for ‘fresh’ products with minimal preservatives and additives results in additional pressures on the industrial microbiologist to discover strategies to meet the consumer demand without endangering the public. This has resulted in the steady development of non-thermal treatments such as microwave and radio frequency, ohmic and inductive heating, high pressure processing, pulsed electrical field and pulsed light, just to name a few that are in development or have been used in commercial food manufacturing (FDA/CFSAN, 2000). These intervention strategies amplify nature’s only controls in controlling these terrorists. For example, high pressure processing uses water pressures to burst the cell. There is a plethora of methods available for the industrial microbiologist to screen and identify this organism. The selection of methods is primarily based on quality and turnaround time. The longer a company’s product takes to reach the market the more it costs the company. Therefore, there is always pressure to find faster methods to screen out negatives. Some of the most common rapid methods are based either on ELISA type tests (BioMerieux VIDAS, TECRA Unique) or PCR (Oxoid’s BAX and Roche’s real-time PCR protocol). These methods are all automated and have the required regulatory approvals. The covert battle between the industrial microbiologist and Listeria is ongoing with no definite exit time. As long as the consumer enjoys the convenience of ready to eat food, Listeria will be waiting to strike; however, the industrial microbiologist will be there to contain, prevent and eliminate any danger to the public.

The Gram Stain

Gram staining is a procedure that microbiologists are taught in their first practical classes. It is the core of microbiology and fundamental to bacterial classification and identification.

The Gram-positive cell retains the crystal violet – iodine complex made in the first steps of the procedure, despite the subsequent washing, decolourising and counterstaining. Gram-negative cells lose the complex and take on the colour of the counterstain.

Gram reactions can sometimes be misleading, giving either a false positive or a false negative result. There are usually three reasons for a false reaction. The culture is not pure, the age of the culture is old or there is a problem with the method applied.

1. Impure Micro-organism

If Gram-positive and Gram-negative cells appear on the same slide, the first step should be to check the purity of the culture. This can be performed by visually looking at the different colony types. Mixed cultures can cause this reaction, however there are some which are variable in their gram reaction.

2. Age of the Culture

Some Gram-positive bacteria appear Gram-negative when they have reached a certain age. This can vary from hours to days.

On the other hand some Gram-negative bacteria become Gram-positive when the culture is quite old. If you suspect this is the case, stain at 2 or 3 different ages and see when the change occurs. Gram reactions should be determined on very young culture, after growth on the plate has become just visible. Some micro-organisms are truly Gram-variable, appearing Gram-positive or Gram-negative according to the conditions.

3. Problems with the Gram Staining Method

If you suspect a problem with the method, check it against a reputable source or text. Here are some hints on performing a correct Graim stain:

ï‚· Greasy slides lead to poor staining, as water solutions run into droplets. Water spreads out in a thin uniform film on slides that are grease free. New slides are generally not clean enough for staining. Slides should be cleaned in alkaline potassium permanganate and then washed with distilled water or 70% alcohol. Avoid touching the slide with fingers by using forceps when handling.

ï‚· When preparing the smear, avoid overcrowding of cells as this prevents proper decolourisation during the washing steps. Cells should lie separately, with approximately 100 cells per microscopic field. A good smear should be not more than barely visible on the slide after staining.

ï‚· Heat fixing smears can sometimes cause Gram-positive cells to stain negatively. If this is the case try methanol fixation; air dry the fresh culture onto the slide, cover with methanol and allow to evaporate at room temperature, then proceed with staining. Gram-positive bacteria fixed in this way are more resistant to decolourisation.

ï‚· Stock solutions of I2 – KI in water are unstable. Store below 25ï‚°C away from light for not longer than 3 weeks. If 12 degrades, Gram-positive bacteria will stain negative. Use freshly prepared solutions where possible or add polyvinylpyrolidine at 1%; this complexes with the 12 and makes it quite stable.

 When washing the slide, don’t run water directly onto the smear. Dip the slide into tap water in a 250mL beaker and have tap water running into the beaker constantly.

 Examine preparations with the oil immersion objective of the bright field microscope, with the condenser fully open. Don’t use phase contrast, as this does not allow recognition of true colours.

ï‚· Weakly Gram-positive bacteria are best detected if the preparation is not counterstained. In this case phase contrast or bright field can be used to differentiate cells.

Gram Stain – A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References This is a 3-in-1 reference book. It gives a complete medical dictionary covering hundreds of terms and expressions relating to gram stain. It also gives extensive lists of bibliographic citations. Finally, it provides information to users on how to update their knowledge using various Internet resources. The book is designed for physicians, medical students preparing for Board examinations, medical researchers, and patients who want to become familiar with research dedicated to gram stain.If your time is valuable, this book is for you. First, you will not waste time searching the Internet while missing a lot of relevant information. Second, the book also saves you time indexing and defining entries. Finally, you will not waste time and money printing hundreds of web pages.

Researchers have revealed that anthrax spores may survive traditional drinking water disinfection methods, according to a Feb. 17 American Society for Microbiology (ASM) press release.

Researchers convened recently at the 2006 ASM Biodefense Research Meeting to determine the fate of anthrax spores in a drinking water system that uses chlorine as a disinfectant, the release said.

According to the statement, the report suggests that water treatment facilities should be prepared to employ alternate disinfection methods (such as exposure to higher concentrations of chlorine or an alternate disinfectant for an extended period of time) in the unlikely event of the release of anthrax into the water supply.

“The data seem to suggest that anthrax spores can tolerate water treatment, can attach to pipes or biofilms within the pipes, and could pass through pipe systems to reach the consumer tap,” Jon Calomiris of the Air Force Research Laboratory at Aberdeen Proving Ground, Edgewood, MD, said in the release.

Source: WaterTechOnline